Fluorescence Probes Used For Detection of Reactive Oxygen Species

J. Biochem. Biophys.
Methods 65 (2005) 45 – 80
www.elsevier.com/locate/jbbm
Review
Fluorescence probes used for detection of reactive

oxygen species
Ana Gomes, Eduarda Fernandes *, José L.F.C. Lima
REQUIMTE, Departamento de Quı́mica-Fı́sica, Faculdade de Farmácia, Universidade do Porto, Rua Anı́bal Cunha,
164, 4099-030 Porto, Portugal
Received 20 April 2005; received in revised form 21 September 2005; accepted 12 October 2005
Abstract
Endogenously produced pro-oxidant reactive species are essential to life, being involved in several
biological functions. However, when overproduced (e.g. due to exogenous stimulation), or when the levels
Abbreviations: ROS, reactive oxygen species; O! ! !

2 , superoxide radical; HO2, hydroperoxyl radical; HO , hydroxyl
! ! 1
radical; ROO , peroxyl radical; RO , alkoxyl radical; H2O2, hydrogen peroxide; O2, singlet oxygen; HOCl,
hypochlorous acid; UV, ultraviolet; RNS, reactive nitrogen species; !NO, nitric oxide; !NO2, nitrogen dioxide radical;
ONOO, peroxynitrite anion; ONOOH, peroxynitrous acid; ONOOCO +
2 , nitrosoperoxycarbonate anion; NO2 , nitronium
cation; N2O3, dinitrogen trioxide; HE, hydroethidine; E+, ethidium; DNA, deoxyribonucleic acid; HPLC, high-
performance liquid chromatography; DPBF, 1,3-diphenylisobenzofuran; SOD, superoxide dismutase; OCl, hypochlorite
anion; PDA, 12-(1-pyrene)dodecanoic acid; DCFH, 2,7-dichlorodihydrofluorescein; DCF, 2,7-dichlorofluorescein;
DCFH-DA, DCFH diacetate form; HRP, horseradish peroxidase; DFC!, DCF’s semiquinone radical; NADH,
nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; GSH, glutathione; K m,
Michaelis–Menten constant; HVA, homovanillic acid; DHR, dihydrorhodamine 123; EDTA, ethylenediaminetetraacetic
acid; DPAX, 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-one; DPA, 9,10-diphenylanthracene;
DPAX-EP, DPAX endoperoxide; EP-1, 3-(4-methyl-1-naphthy)propionic acid endoperoxide; DMA, 9,10-dimethylan-
thracene; DMA-EP, DMA endoperoxide; DMAX, 9-[2-(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one;
DMAX-EP, DMAX endoperoxide; !CH3, methyl radical; CHD, 1,3-cyclohexanedione; 7-OHC, 7-hydroxycoumarin;
3-CCA, coumarin-3-carboxylic acid; SECCA, 3-CCA’s succinimidyl ester; HPF, 2-[6-(4V-hydroxy)phenoxy-3H-xanthen-
3-on-9-yl]benzoic acid; APF, 2-[6-(4V-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid; MPO, myeloperoxidase;
FL, fluorescein; HORAC, hydroxyl radical averting capacity; cis-PnA, cis-parinaric acid; C11-BODIPY581/591,
4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid; AMVN, 2,2V-azobis-2,4-
dimethylvaleronitrile; AAPH, 2,2V-azobis(2-amidinopropane) dihydrochloride; C11-fluor, 5-(N-dodecanoyl)aminofluor-
escein; fluor-DHPE, dihexadecanoylglycero-phosphoethanolamine; DPPP, diphenyl-1-pyrenylphosphine; DPPPjO,
diphenyl-1-pyrenylphosphine oxide; PMNs, polymorphonuclear leukocytes; PMA, phorbol 12-myristate 13-acetate;
DCFH-DA, 2,7-Dichlorodihydrofluorescein diacetate; DCF, 2,7-dichlorofluorescein; TRAP, total peroxyl radical
trapping potential; ORAC, oxygen radical absorbance capacity; AUC, area under curve; RMCD, randomly methylated
h-cyclodextrins.
* Corresponding author. Tel.: +351 222078968; fax: +351 222004427.
0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbbm.2005.10.003
46 A. Gomes et al. / J. Biochem. Biophys. Methods 65 (2005) 45–80
of antioxidants become severely depleted, these reactive species become highly harmful, causing oxidative
stress through the oxidation of biomolecules, leading to cellular damage that may become irreversible and
cause cell death. The scientific research in the field of reactive oxygen species (ROS) associated biological
functions and/or deleterious effects is continuously requiring new sensitive and specific tools in order to
enable a deeper insight on its action mechanisms. However, reactive species present some characteristics
that make them difficult to detect, namely their very short lifetime and the variety of antioxidants existing in
vivo, capable of capturing these reactive species. It is, therefore, essential to develop methodologies capable
of overcoming this type of obstacles. Fluorescent probes are excellent sensors of ROS due to their high
sensitivity, simplicity in data collection, and high spatial resolution in microscopic imaging techniques.
Hence, the main goal of the present paper is to review the fluorescence methodologies that have been used
for detecting ROS in biological and non-biological media.
D 2005 Elsevier B.V. All rights reserved.
Keywords: Fluorescence probe; Reactive oxygen species; Free radical; Antioxidant; Oxidative stress; Scavenging activity
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2. Fluorescence probes for detection of superoxide radical . . . . . . . . . . . . . . . . . . . 49
2.1. Hydroethidine (dihydroethidium; HE) . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.2. 1,3-Diphenylisobenzofuran (DPBF) . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.3. 2-(2-Pyridil)-benzothiazoline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
3. Fluorescence probes for detection of hydrogen peroxide . . . . . . . . . . . . . . . . . . . 52
3.1. 2,7-Dichlorodihydrofluorescein (DCFH) . . . . . . . . . . . . . . . . . . . . . . . . 52
3.2. Scopoletin (7-hydroxy-6-methoxy-coumarin) . . . . . . . . . . . . . . . . . . . . . . 54
3.3. N-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) . . . . . . . . . . . . . . . . . . 55
3.4. Homovanillic acid (4-hydroxy-3-methoxy-phenylacetic acid; HVA) . . . . . . . . . . 57
3.5. Dihydrorhodamine 123 (DHR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
4. Fluorescence probes for detection of singlet oxygen . . . . . . . . . . . . . . . . . . . . . 59
4.1. 9,10-Dimethylanthracene (DMA). . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.2. 9-[2-(3-Carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones
(DPAXs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
4.3. 9-[2-(3-Carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one
(DMAX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
5. Fluorescence probes for detection of hydroxyl radical . . . . . . . . . . . . . . . . . . . . 61
5.1. 4-(9-Anthroyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl . . . . . . . . . . . . . . . . 61
5.2. 1,3-Cyclohexanedione (CHD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.3. Sodium terephthalate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
5.4. Coumarin, coumarin-3-carboxylic acid (3-CCA) and N-succinimidyl
ester of coumarin-3-carboxylic acid (SECCA) . . . . . . . . . . . . . . . . . . . . . 64
5.5. 2-[6-(4V-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF)
and 2-[6-(4V-amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF). . . . . . . . 65
5.6. Fluorescein (FL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6. Fluorescence probes for detection of peroxyl radical . . . . . . . . . . . . . . . . . . . . . 66
6.1. cis-Parinaric acid (cis-PnA, (18:14):9,11,13,15-cis-trans-trans-cis-
octadecaenoic acid). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
6.2. 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-
3-undecanoic acid (C11-BODIPY581/591) . . . . . . . . . . . . . . . . . . . . . . . . 67
6.3. Lipophilic fluorescein derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.4. Dipyridamole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
A. Gomes et al. / J. Biochem. Biophys. Methods 65 (2005) 45–80 47
6.5. Diphenyl-1-pyrenylphosphine (DPPP) . . . . . . . . . . . . . . . . . . . . . . . . . 71

6.6. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) . . . . . . . . . . . . . . . . . 72
6.7. h-Phycoerythrin/Fluorescein/6-Carboxyfluorescein . . . . . . . . . . . . . . . . . . . 73
7. Final comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
1. Introduction
Reactive oxygen species (ROS), according to their own name, derive and present higher
reactivity than molecular oxygen with redox activity [1,2]. The ROS designation comprehends
not only free radicals, such as superoxide radical (O2!), hydroperoxyl radical (HO2!), hydroxyl
radical (HO!), peroxyl radical (ROO!) and alkoxyl radical (RO!), but also non-radicals, namely
hydrogen peroxide (H2O2), singlet oxygen (1O2), and hypochlorous acid (HOCl) [2,3]. By
definition, free radicals are atoms or molecules, capable of independent existence, that possess
one or more unpaired electrons [4]. Electrons are more stable when paired together in orbitals:
the two electrons in a pair have different directions of spin. Hence, radicals are generally less
stable than non-radicals, although their reactivity varies.
It is well known that the exposition to certain noxious factors, such as some xenobiotics,
infectious agents, pollution, UV light, cigarette smoke and radiation, may lead to the production
of ROS [5,6]. On the other hand, ROS, as well as reactive nitrogen species (RNS) like nitric
oxide (!NO) nitrogen dioxide radical (!NO2), but also non-radicals, like peroxynitrite anion
(ONOO), peroxynitrous acid (ONOOH), nitrosoperoxycarbonate anion (ONOOCO2),
nitronium cation (NO2+), and dinitrogen trioxide (N2O3), are continuously generated in small
quantities on normal cellular processes. Endogenously produced ROS and RNS are essential to
life, being involved in different biological functions, namely signal transduction, neurotrans-
mission, smooth muscle relaxation, peristalsis, platelet aggregation, blood pressure modulation,
immune system control, learning and memory, production of energy, fagocytosis, the regulation
of cellular growth, cellular signalling, the synthesis of important biological compounds and the
metabolism of xenobiotics [7–10]. However, when overproduced, or when the levels of
antioxidants become severely depleted, these reactive species become highly harmful, causing
oxidative stress through the oxidation of biomolecules, such as the lipids of cellular membranes,
tissue proteins or enzymes, carbon hydrates and DNA, leading to cellular damage that becomes
irreversible at a certain point. It is of extreme importance the fact that oxidative stress has been
implicated in the aetiology of several diseases and in aging [11–16]. Consequently, in a normal
cellular environment, ROS are essential to life, while in case of overproduction or exhaustion of
antioxidants they might become deleterious.
The scientific research in the field of ROS associated biological functions and/or deleterious
effects is continuously requiring new sensitive and specific tools that may enable a deeper
insight on its action mechanisms. However, reactive species present some characteristics that
make them difficult to detect, namely their very short lifetime and the variety of antioxidants
existing in vivo, capable of capturing these reactive species. It is, therefore, essential to develop
methodologies capable of overcoming this type of obstacles. The fluorescence methodology,
associated with the use of suitable probes, is an excellent approach to measure ROS because of
its high sensitivity, simplicity in data collection, and high spatial resolution in microscopic
imaging techniques [17,18].
Table 1
Summary of the detected ROS, excitation/emission wavelengths, reactant induced fluorescence changes and main
applications for the probes reported in the review
Probe ROS detected Excitation/ Reactant induced Main reported
emission fluorescence changes applications
wavelengths
(nm)
HE O!
2 520/610 Production of a red Useful for monitoring the
fluorescent compound oxidative burst in cells
(probably ethidium)
1
DPBF O!
2 410/455 Fluorescence decrease O2 and O! 2 detection in
1
O2 or 477 phospholipid liposomes
2-(2-Pyridil)- O!
2 377/528 Production of the fluorescent Determination of SOD
benzothiazoline 2-(2-pyridil)-benzothiazol activity
DCFH H2O2 498/522 Production of the fluorescent The diacetate form
HO! 2,7-dichlorofluorescein (DCFH-DA) can be applied
ROO! in cell studies. Useful as
a marker of the cellular
oxidative stress
Scopoletin H2O2/HRP 360/460 Fluorescence decrease due Widely used as an H2O2
to scopoletin’s oxidation monitoring probe, either in
isolated mitochondria, or in
stimulated neutrophils and
eosinophils
Amplex Red H2O2/HRP 563/587 Production of a highly Detection of H2O2 in
fluorescent product: activated
resorufin phagocytic cells as well as in
other types of cells or in
non-cellular systems
HVA H2O2/HRP 312/420 Production of a fluorescent Useful for detecting the
dimer production of H2O2 in
isolated mitochondria of
various tissues
DHR H2O2/HRP 505/529 Production of the fluorescent H2O2, HOCl and ONOO
HOCl rhodamine 123 detection in cells. Evaluation
of scavenging activity
1
DMA O2 375/436 Fluorescence decrease by Specific 1O2 detection
generation of a non-
fluorescent endoperoxide
1
DPAX O2 495/515 Production of a fluorescent Specific 1O2 detection
endoperoxide
1
DMAX O2 495/515 Production of a fluorescent Specific 1O2 detection.
endoperoxide Potential usefulness for
assays in biological systems
4-(9-Anthroyloxy)- HO! 377/427 Fluorescence increase Indirect detection of HO!,
2,2,6,6- through the elimination of involving its reaction with
tetramethylpiperidine- the intramolecular quenching DMSO and !CH3 production
1-oxyl
CHD HO! 400/452 Production of a fluorescent Indirect detection of HO!,
compound based on its reaction with
DMSO
Sodium HO! 310/430 Production of the fluorescent Specific HO! detection
terephthalate sodium 2-hydroxy- (involves hydroxylation
terephthalate reactions)
Table 1 (continued)
Probe ROS detected Excitation/ Reactant induced Main reported
emission fluorescence changes applications
wavelengths
(nm)
3-CCA and HO! 350 and Generation of fluorescent Screening of HO! scavenging
SECCA 395/450 hydroxylated coumarins activity and HO! generation
activity
HPF and APF HO! 500/520 Generation of the fluorescent Detection of HO! or HOCl
HOCl fluorescein by O-dearylation (by combination of the two
(only APF) probes) in either cellular or
non-cellular systems
cis-PnA ROO! 320/432 Irreversible fluorescence Evaluation of lipid
loss upon oxidation peroxidation in different
types of cells
C11-BODIPY581/591 ROO! 510/595 Alteration in fluorescence Evaluation of lipid
RO! properties (turns from red peroxidation and antioxidant
HO! to green) efficacy in model
membranes, lipoproteins,
biological fluid
and living cells
Lipophilic fluorescein ROO! 495/515 Fluorescence quenching Determination of lipid
derivatives by hydroperoxides peroxidation associated to
the cellular membrane
Dipyridamole HO! 415/480 Fluorescence decay upon Assessment of the activity of
O!
2 oxidation hydrosoluble or liposoluble
ROO! antioxidants
DPPP ROO! 351/380 Generation of a fluorescent Measurement of the extent of
product: DPPPO oxidation in solution and in
low-density lipoprotein
particles. Evaluation of lipid
peroxidation in living cell
membranes
h-Phycoerythrin ROO! 520/580 Fluorescence decay upon Evaluation of the antioxidant
oxidation capacity
Fluorescein/ ROO! 495/515 Fluorescence decay upon Evaluation of the antioxidant
6-carboxyfluorescein oxidation capacity. Screening of ROO!
scavenging activity
The main goal of this review is to present the fluorescence methodologies that have been used
for detecting ROS in biological and non-biological environments. A special emphasis will be
given to the advantages and limitations of the different methodologies that have been developed
for that effect. A summary of the detected ROS, excitation/emission wavelengths, reactant
induced fluorescence changes and main applications for the probes reported in the review is
given in Table 1.
2. Fluorescence probes for detection of superoxide radical
2.1. Hydroethidine (dihydroethidium; HE)
Hydroethidine (dihydroethidium; HE) has been used as a fluorescent probe for detecting O2!
due to its reported relative specificity for this ROS [19–22]. Indeed, when HE is oxidized by
O2!, it originates ethidium (E+) a fluorescent compound (k excitation = 520 nm; k emission = 610 nm)
(Fig. 1) [23]. However, Tarpey et al. [24] pointed out some limitations in the use of HE when
detecting or quantifying O2!. Firstly cytochrome c is able to oxidize HE, an aspect that might be
important in situations where the main source of O2! is mitochondria or in situations where
cytochrome c is released to cytosol during apoptotic processes. Due to the interconnection
between oxidative stress and the apoptotic processes, it will be difficult, in these situations, to
assume that the HE oxidation to E+ only results from the action of O2!. Secondly, the use of high
HE concentrations might lead to a fluorescence increase independent of O2! as result of a
formation of E+ that exceeds the connection capacity of the mitochondrial nucleic acids,
allowing its connection to nuclear DNA, thus causing a substantial fluorescence increase.
Thirdly, the O2! quantification might not be exact by this method because HE increases the O2!
dismutation rate to H2O2 [20,24].
Importantly, HE can be also oxidized by H2O2 via non-specific peroxidase (horseradish
peroxidase and myeloperoxidase) catalysis, forming fluorescent oxidation products [25]. These
products give excitation/emission peaks (490–495/580–600 nm) near the excitation/emission
peaks (475/580 nm) of the HE-superoxide oxidation product, and this may pose serious
interference problems to the fluorescent detection of the O2!. Furthermore, HE can also be
oxidized by a variety of reactive species, being the relative reactivities ranked by
ONOO N Fe(II)/H2O2 (i.e. HO!) N O2! N H2O2. Thus, in fact, HE provides an index of ROS
and RNS production [2].
HE has the ability to cross-cellular membranes becoming therefore useful in assessment
studies for the oxidative burst in cells [19,22,26–28]. Inside the cell HE is oxidized to E+, which
in its turn is retained in the nucleus, mixing itself with the DNA, a fact that increases its
fluorescence [20,22,24,26,29].
Zhao et al. [29] have recently considered the possibility of not being E+ the oxidation product
of HE by O2!. Fluorescence, high-performance liquid chromatography (HPLC) and HPLC-mass
spectrometry studies, carried out by this group of researchers, indicated that O2! reacts with HE
originating a fluorescent product distinct from E+. This product presents a different molecular
weight and an emission maximum at 567 nm whereas the emission maximum of the E+ is at 610
nm. However, the chemical structure of this product is yet to be determined [29].
Zhao et al. [29] have verified that in the presence of other ROS and RNS (ONOO, HO!,
H2O2) HE does not originate the same oxidation product observed with the O2!. According to
Zhao et al. [29], their discoveries can lead to the development of better fluorescence
H2N NH2 H2N NH2

+
H N N
CH2CH3 CH2CH3
HE E+
λexcitation = 520 nm
λemission = 610 nm
Fig. 1. Chemical structure of hydroethidine (HE) and ethidium (E+).

methodologies for detecting O2!. Nevertheless, more studies are clearly needed in order to
evaluate the specificity of the reaction between HE and O2! in biological systems.
The observation that various concentrations of cytochrome c can accept one to four electrons
per mole HE raised the possibility that the octahedrally coordinated heme Fe(III) of other proteins
may act as non-specific electron donor to HE [30]. Possible protein oxidants of HE with heme
Fe(III) in octahedral coordination bonding are the cytochromes of mitochondrial, photosynthetic,
microsomal, and bacterial electron transport chains and the globins hemoglobin and myoglobin.
Indeed, these authors showed that HE can react non-enzymatically with the heme Fe(III) of
mitochondrial cytochromes c, c1, b566, b562, and aa3 in a O2 independent way, while that with
the heme Fe(III) of hemoglobin-Fe(III) (metHb) and myoglobin-Fe(III) (metMb) was strictly O2
dependent. HE is also expected to react with photosystem II cytochromes b6 and f (c-type
cytochrome) and with the microsomal cytochrome b5 (and possibly cytochrome P450) [30].
2.2. 1,3-Diphenylisobenzofuran (DPBF)
1,3-Diphenylisobenzofuran (DPBF) is a molecule which, under certain conditions, presents

fluorescence, namely when incorporated in phospholipid liposomes (k excitation = 410 nm;
k emission = 455 or 477 nm) (Fig. 2) [31].
In 1999, it was demonstrated by Ohyashiki et al. [31] that it was possible to use DPBF in the
detection of O2! in phospholipid liposomes. These investigators observed that the fluorescence
of this probe, incorporated in liposomes, decreases throughout time in the presence of xanthine/
xanthine oxidase. This fluorescence decrease was suppressed by the addition of superoxide
dismutase (SOD). In its turn, the addition of catalase did not have any protection effect against
the fluorescence decrease. Also, the addition of H2O2 to the liposomes marked with DPBF did
not cause any change on the fluorescence of the probe. They concluded that the fluorescence
decrease was due to the reaction of DPBF with O2!, although the mechanism of this reaction
was not proposed. The possibility of HO! or 1O2 being responsible for the fluorescence decrease,
when the xanthine/xanthine oxidase generation system for O2! is used, was also excluded by the
use of specific scavengers for these reactive species, which did not had any protective effect on
the fluorescence quenching [31].
However, it was previously demonstrated that the DPBF/liposomes fluorescence can be
quenched by 1O2 specific generating systems. This was shown for 1O2 generation by OCl/H2O2
(neither OCl, nor H2O2 directly quenched DPBF/liposomes fluorescence) [32], photoirradia-
tion of hematoporphyrin [33], photoirradiation of erythrosine [34], and photoirradiation of water
soluble methyleneblue or lipid soluble 12-(1-pyrene)dodecanoic acid (PDA) [35]. Thus, DPBF
DPBF
λemission = 455 or 477 nm
Fig. 2. Chemical structure of 1,3-diphenylisobenzofuran (DPBF).

can be useful as a fluorescent indicator either for monitoring O2! or 1O2 in phospholipid
liposomes.
2.3. 2-(2-Pyridil)-benzothiazoline
2-(2-Pyridil)-benzothiazoline was synthesized by Tang et al. [36] with the objective of being
applied for detecting O2! and in the determination of the SOD activity by flow injection analysis
with spectrofluorimetric detection. In this study O2! was generated through the following
reaction (1) [36]:

S2 O2 2 !
4 þ O2 þ 4HO Y 2SO3 þ 2H2 O þ O2 ð1Þ
2-(2-Pyridil)-benzothiazoline (non-fluorescent), by reaction with O2!,
originates a highly
fluorescent product, 2-(2-pyridil)-benzothiazol (k excitation = 377 nm; k emission = 528 nm) (Fig. 3).
The use of another O2! generation system (auto-oxidation of pyrogallol), as well as of SOD,
reinforced the theory that it is the O2! species responsible for the oxidation of the probe [36].
The ideal pH interval for this methodology is between 8.9 and 10.0 units due to the generation
system used, which only produces O2! under alkaline conditions [36].
H2O2 is not able to oxidize the probe, and HO!, in the same concentration, does not cause a
fluorescence increase [36].
3. Fluorescence probes for detection of hydrogen peroxide
3.1. 2,7-Dichlorodihydrofluorescein (DCFH)
The oxidation of 2,7-dichlorodihydrofluorescein (DCFH) originates 2,7-dichlorofluorescein

(DCF) (Fig. 4), a fluorescent compound (k excitation = 498 nm; k emission = 522 nm) initially thought
to be useful as a specific indicator for H2O2 [37]. However, it was already demonstrated that
DCFH is oxidized by other ROS, such as HO! and ROO! (see below in the peroxyl radical
section), and also by RNS like !NO and ONOO [38,39].
The DCFH diacetate form (DCFH-DA) (Fig. 4) can be applied in cell studies due to its ability
to diffuse through the cellular membrane, being then enzymatically hydrolysed by intracellular
esterases to DCFH [39,40].
The presence of cellular peroxidases is important for the oxidation of DCFH to DCF by H2O2
[38,41]. Of note, horseradish peroxidase (HRP) is also capable of oxidizing DCFH even in the
absence of H2O2 [24,38]. These observations for HRP led to the hypothesis that oxidation of
DCFH could also be directly performed by other oxidases or peroxidases. Zhu et al. [42] and
Hempel et al. [43] reported that DCFH is oxidized by xanthine oxidase. However, this report is
O2.- N
HN
N S N S
2-(2-Pyridil)-benzothiazoline 2-(2-Pyridil)-benzothiazol
λemission = 528 nm
Fig. 3. Oxidation of 2-(2-pyridil)-benzothiazoline by O!

2 to 2-(2-pyridil)-benzothiazol (adapted from Ref. [36]).
HO O OH HO O O
Cl Cl Cl Cl
H Oxidation
COOH COOH
DCFH DCF
(Non fluorescent) (Fluorescent)
Esterase
λemission = 522 nm
or HO-
OCOCH 3 O OCOCH3
Cl Cl
H
COOH
DCFH-DA
(Non fluorescent)
Fig. 4. Mechanism of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) de-esterification to 2,7-dichlorodihydro-

fluorescein (DCFH), and further oxidation to fluorescent 2,7-dichlorofluorescein (DCF) by ROS and RNS (adapted from
Ref. [38]).
in contradiction with other observations [38]. Hempel et al. [43] also indicated the ability that
catalase and SOD have to oxidize the DCFH. Besides cellular peroxidases, hematin and
cytochrome c are substances that highly increase the formation of DCF [24, 44]. Noteworthy,
Lawrence et al. [45] demonstrated that cytochrome c is a powerful catalyst of DCFH oxidation,
and so use of DCFH-DA to probe oxidative stress during apoptosis should be approached with
caution, since a rise in cytosolic cytochrome c levels could result in a higher fluorescence
without any change in cellular peroxide levels.
DCFH oxidation also occurs by action of H2O2 in presence of Fe(II) [26,44], being,
nevertheless, highly probable that, in this case, HO! is the species responsible for the oxidation
[41,42]. There are several evidences that O2! is not capable of oxidizing DCFH [38,42]. Myhre
et al. [41] also classified it as non-appropriate for determining O2!, HOCl and !NO. On the other
hand, these authors considered DCFH as a sensible probe, not only for H2O2 in presence of
cellular peroxidases, but also for the determination of ONOO, and HO!.
DCF can suffer photoreduction whether in presence of visible light, or by action of UVA
radiation [46]. This reduction’s mechanism is supposed to involve the generation of semiquinone
radical from DCF (DFC!) which, by reaction with O2, originates O2!. In its turn, the
dismutation of O2! generates H2O2, which leads to an artificial increase of DCFH oxidation
and consequently to an amplification of DCF fluorescence. DFC! is formed not only
photochemically but can also be enzymatically catalysed by the pair peroxidase/H2O2 (Fig. 5).
This is a factor to be taken into account in tests where the fluorescence of DCF depends of
peroxidases and in tests aiming to study the formation of ROS in UVA irradiated cells [46,47].
Due to the existence of several substances that interfere with the formation of DCF, this
probe, when used in cellular systems, has better use as a marker of the cellular oxidative stress
HO O OH
OCOCH3 O OCOCH3
Esterase
or HO-
Cl Cl
Cl Cl H
H COO-
COO-
DCFH-DA DCFH
O2
Auto-oxidation
H2O2/Peroxidase
O2.- O2
HO O O HO O OH
1,3(DCF)* -H+
Cl Cl hv
Cl . Cl
COO- DCFH DCF.- COO -
DCF DCF.-
Fig. 5. Mechanism of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) de-esterification to 2,7-dichlorodihydro-

fluorescein (DCFH) and further conversion into DCF! (adapted from Ref. [46]).
than as indicator of the formation of H2O2 or other ROS and RNS [24,48]. In fact DCFH has
been used as an oxidative burst indicator in macrophages and neutrophil [49,50] and also as a
way of studying the production of oxidizing substances by action of multiple stimulus in other
kinds of cells [51,52].
3.2. Scopoletin (7-hydroxy-6-methoxy-coumarin)
Scopoletin (7-hydroxy-6-methoxy-coumarin) (Fig. 6) is a naturally occurring fluorescent

compound (k excitation = 360 nm; k emission = 460 nm). H2O2, in presence of HRP, or catalase, forms
a complex, which oxidizes scopoletin, originating a non-fluorescent product (an inverse
fluorescence measurement) [53–56]. Aqueous solutions of scopoletin were found to be
reasonably stable in diffused light and not oxidized by either peroxidases or H2O2 alone [54].
Although being widely used as an H2O2 monitoring probe, either in isolated mito-
chondria, or in stimulated neutrophils and eosinophils [55–59], scopoletin has some
disadvantages: i) has low extinction coefficient; ii) presents an excitation and emission
short wavelength spectra which makes it susceptible to interference from autofluorescence
when biological samples are used; iii) due to its low fluorescent power scopoletin requires a
significant signal amplification to quantify its fluorescence changes, which results in an
MeO
HO O O
Scopoletin
λexcitation= 360 nm
λemission = 460 nm
Fig. 6. Chemical structure of scopoletin.
increase of the background fluorescence therefore affecting the method’s sensibility; iv)
presents low stability in biological media. With time its fluorescence decreases spontaneously
and is highly dependent on pH and temperature; v) might suffer interference from reductive
compounds such as NADPH, NADH, ascorbic acid and glutathione (GSH); vi) the pair
scopoletin/HRP is problematic for studies with eosinophils since reagent induced cell
activation occurs [54–56,58,60].
3.3. N-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red)
N-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) is a non-fluorescent molecule that when

oxidized by H2O2 in presence of HRP originates resorufin, a highly fluorescent product
(k excitation = 563 nm; k emission = 587 nm) (Fig. 7) [55,56]. Amplex Red consumes stoichiometric
amounts of H2O2. This probe can also be used for measurement of O2!, by using SOD in the
reaction mixture to convert this radical into H2O2 [61]. As its advantages Amplex Red presents
low background fluorescence (this probe does not present fluorescence, only its oxidation
product) as well as stability and high fluorescence power of its oxidation product, all this
resulting in a sensibility increase for detecting H2O2. Furthermore, this probe’s excitation and
emission maximum wavelengths subsist in a spectral zone that has little susceptibility to
interference from autofluorescence in assays where biological samples are used [55,56].
However, resorufin per se is also a substrate of HRP. Further oxidation of resorufin, to non-
fluorescent resazurin and other oxidation products (Fig. 7), may result in significant loss of
fluorescence intensity [55,62].
The high affinity of hydrogen peroxide to the active site of HRP was demonstrated with a low
Michaelis–Menten K m value for H2O2, which was reported to be 1.55 AM when using Amplex
Red as the reductant substrate. Nevertheless, increasing the H2O2 concentration beyond four
times the K m values beyond ~6.0 AM would not substantially increase the rate of the intended
enzymatic reaction transformation of Amplex Red to resorufin, since it would significantly
enhance the HRP inhibition and suicide inactivation by an excess amount of H2O2 at the active
site by forming inactive form of HRP-reversible Compound III and irreversible P670 (k max = 670
nm) [62].
In the absence of HRP and H2O2, discernible changes in the fluorescence intensity of
resorufin were also observed by simply dissolving resorufin in aqueous solutions in the pH range
6.2–7.7, near it’s pKa value [62]. In addition, the stability of Amplex Red may be an issue at
high pH values (N 8.3), since de-N-acetylation could occur via nucleophilic substitution and
general base catalysis, resulting in labile dihydroxyphenoxazines [62].
O
COCH3
2H2O2 2H 2O + O2
N N N
HRP 3
1 HO O O
HO O OH HO O O
Amplex Red Resorufin Resazurin

(Non fluorescent) (Highly fluorescent) (Non fluorescent)
λemission = 587 nm
Unknown
Fluorescent
Fig. 7. Reaction scheme for the horseradish peroxidase (HRP)-catalyzed Amplex Red oxidation by H2O2 (adapted from
Ref. [62]).
Mohanty et al. [55] carried out tests where catalase was used in order to confirm that the
observed fluorescence resulted indeed from action of H2O2. In these tests they verified that the
fluorescence decreased significantly when catalase was added to the reaction mixture after the
other reagents and that catalase totally prevented fluorescence developing when added before
Amplex Red. Another evidence that this probe presents specificity for H2O2 is that the
hypoxanthine/xanthine oxidase system generated O2! did not produce the probe’s fluorescence
increase. HOCl caused an increase on the probe’s fluorescence but only in very high
concentrations (z 1 mM) [55].
Thus, Amplex Red is a probe sensible and specific for the detection of H2O2, and can be used
not only in activated phagocytic cells but also in other types of cells or even in non-cellular
systems [55,56]. The sensitivity of the Amplex Red when detecting H2O2 is at least 10 times
higher than scopoletin’s under the same conditions [55]. Another study where the two mentioned
probes were compared revealed a sensibility to H2O2 5 to 20 times higher to Amplex Red in
relation to scopoletin [56]. Zhou et al. [56] referred a 50 nM detection limit for H2O2 in a plate
reader methodology in which Amplex Red was used as probe. Nevertheless, it must be taken
into account that in biological systems, NADH and GSH may interfere with HRP/Amplex Red
assay system. Indeed, auto-oxidation and HRP-mediated oxidation of NADH and GSH may
produce H2O2 at levels found in biological systems [63]. Also, this methodology, like every
other HRP dependent methodologies, is susceptible to interference from substances that oxidize
this enzyme. Another aspect to be considered in this methodology is that Amplex Red seems to
be a substrate for endogenous peroxidases present in eosinophils and in neutrophils, which can
also cause interference [55].
According to Towne et al. [62], the following aspects must be taken into consideration
to obtain reliable quantitative results when using Amplex Red for measuring H2O2 or
H2O2-generating systems: (i) The pH range of the assay should be kept between 7.5 and
8.5 for fluorescence stability and assay sensitivity; (ii) The H2O2 concentration range must
be kept between 0.01 and 100 AM using a logistic four-parameter equation for curve
fitting [64] to account for the plateau region at higher H2O2 concentrations. This range
may need to be narrowed when using linear fit for data analysis due to deviation from
linearity in the fluorescence intensity vs. [H2O2] plot; (iii) The incubation time should be
kept as short as possible to minimize the contributions from reactions other than the
intended reaction 1 shown in Fig. 7; (iv) At high H2O2 concentrations, the HRP
concentration should be lowered whenever possible to slow reaction 3, the dominant
pathway leading to lower fluorescent intensity. Indeed, the oxidation product of Amplex
Red, resorufin, can be further oxidized to a non-fluorescent compound. However this
further oxidation will not occur significantly unless the H2O2 concentration is higher than
the Amplex Red concentration in the reaction mixture [54]. Without taking into account
the details of the redox reactions involved with the Amplex Red assay and their potential
impact on assay results, misleading results could be obtained with improperly designed
assay conditions. This might be particularly the case if Amplex Red was used in an assay
with prolonged incubation and/or in the presence of cultured cells, in which pH drift may
occur during the assay.
3.4. Homovanillic acid (4-hydroxy-3-methoxy-phenylacetic acid; HVA)
Homovanillic acid (4-hydroxy-3-methoxy-phenylacetic acid; HVA) represents an important

metabolite of dopamine in the brain. It is a non-fluorescent molecule that by reaction with
H2O2, in presence of HRP, originates a fluorescent dimer (k excitation = 312 nm; k emission = 420
nm) (Fig. 8) [59].
When properly used, this method allows the quantification of the fast and basal production
rates of ROS in biological systems. Indeed, this fluorescent probe has been used for detecting the
production of H2O2 in isolated mitochondria of various tissues, healthy, or otherwise affected by
degenerative diseases [59].
Staniek and Nohl [57] have recently suggested an alternative H2O2 detection method in
mitochondria where the detection system (HVA or scopoletin) does not contact directly with the
mitochondria. The reactional environment that contains mitochondria is subject to a
centrifugation after which the supernatant (containing H2O2) is separated from the mitochondria
and only then has contact with the detection system. According to these authors, this
COOH COOH
COOH
CH2 CH2 CH2
H2O2
HRP MeO OMe
MeO
OH OH OH
Homovanillic acid Fluorescent dimer

(Non fluorescent)
λemission = 420 nm
Fig. 8. Oxidation of homovanillic acid by H2O2 in the presence of horseradish peroxidase (HRP) to a fluorescent dimer
(adapted from Ref. [65]).
methodology allows the elimination of interference caused by the presence of mitochondria

during the monitoring of H2O2. Indeed, HVA, due to its emission wavelength, suffers interference
from mitochondria constituents that are in resonance with this wavelength. Scopoletin in its turn
presents an emission maximum that coincides with the absorption maximum of flavoproteins,
which match with many of the compounds involved in the mitochondrial respiratory chain.
Under these conditions the scopoletin’s fluorescence quenching might occur, in a H2O2 non-
dependent form [57]. According to Staniek and Nohl [57] this methodology allows not only to
exclude the referred interference but also to prevent any possible interaction between the probes
and the different oxidation/reduction systems of the respiratory chain.
Recently, HVA has also been used for the assessment of the different compound’s antioxidant
activity. The presence of substances with H2O2 scavenging activity prevents HVA oxidation by
the removal of H2O2 and prevents the fluorescence increase in a concentration dependent
manner [65]. HVA has the advantage of being specific to H2O2, contrary to DCFH that is
oxidized by other cellular oxidants [59]. Furthermore it is more stable and more sensible than
scopoletin. Its specificity can be assessed through the inhibition of the reaction after the addition
of catalase [66].
HVA has also been used for the fluorimetric detection of peroxidase activity in biochemical
and biological assays. However, other systems like cytochrome c/H2O2, lipoxygenase/H2O2 and
Fenton systems are also able to oxidize HVA to its dimer, thus contributing to possible
misleading results [67]. HVA can be successfully used in the measurement of enzyme activity,
when H2O2 is one of the final products. This has been shown in the measurement of lysyl
oxidase [68], acyl-CoA oxidases [69, 70] and diamine oxidase [71].
3.5. Dihydrorhodamine 123 (DHR)
Dihydrorhodamine 123 (DHR) is a non-fluorescent molecule that, by oxidation, yields

rhodamine 123, a fluorescent cationic and lipophilic probe (k excitation = 505 nm; k emission = 529
nm) (Fig. 9) [72]. The lipophilicity of DHR facilitates its diffusion across cell membranes. Upon
oxidation of DHR to the fluorescent rhodamine 123, one of the two equivalent amino groups
tautomerizes into an imino, effectively trapping rhodamine 123 within cells [38].
H2O2 oxidizes DHR in the presence of peroxidases, although this probe has low specificity
for this ROS since it can also be oxidized by other reactive oxidants, namely ONOO, Fe(II),
Fe(III)/ascorbate, Fe(III)/EDTA, cytochrome c, or HOCl [38,43,44,73]. On the other hand, DHR
+
H2N O NH2 H2N O NH2
Oxidation
H
COOCH3 COOCH3
Dihydrorhodamine 123 Rhodamine 123

(Non fluorescent) (Fluorescent)
λemission = 529 nm
Fig. 9. Oxidation of dihydrorhodamine 123 to rhodamine 123 (adapted from Ref. [38]).
is not directly oxidizable by H2O2 alone, by O2!, and by xanthine/xanthine oxidase

[38,44,72,73].
4. Fluorescence probes for detection of singlet oxygen
4.1. 9,10-Dimethylanthracene (DMA)
9,10-Dimethylanthracene (DMA) is a fluorescent compound (k excitation = 375 nm, k emission =

436 nm) that reacts selectively with 1O2 to form the non-fluorescent 9,10-endoperoxide (Fig. 10)
with a very high rate constant (2 107–9 108 M 1 s 1) in many organic solvents, as well as
water [74–77]. Lavi et al. [78] demonstrated that DMA is not located in a preferential depth in the
membrane, unlike its ionic analogue, 9-anthracenepropionic acid, which anchors at the lipid/water
interface with its charged carboxyl group.
4.2. 9-[2-(3-Carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs)
The most widely used 1O2 trap is 9,10-diphenylanthracene (DPA), which reacts rapidly and
specifically with 1O2 to form a thermostable endoperoxide at a rate of k = 1.3 106 M 1 s 1.
The decrease in absorbance at 355 nm is used as a measure of the formation of the endoperoxide.
However, DPA derivatives are not very sensitive probes because the detection is based on the
measurement of absorbance [79].
Umezaka et al. [79] fused DPA with a fluorophore (fluorescein) aiming to associate the first’s
reactive characteristics with the second’s fluorescent characteristics. Fluorescein was chosen as
fluorophore since it has a high fluorescence quantum yield in aqueous solution and is able to be
excited at long wavelength. From this fusion resulted 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-
hydroxy-3H-xanthen-3-ones (DPAXs) (Fig. 11) [79]. Thus, DPAXs were the first chemical traps
for 1O2 that permitted fluorescence detection. They react with 1O2 to produce DPAX
endoperoxides (DPAX-EPs) (Fig. 11). DPAXs themselves scarcely fluoresce, while DPAX-
EPs are strongly fluorescent. The mechanism accounting for the diminution of fluorescence in
DPAXs and its enhancement in DPAX-EPs remain unclear [79].
The fluorescence intensity of fluorescein derivatives is known to be decreased under acidic
conditions as a consequence of the protonation of the phenoxide oxygen atom. In order to
stabilize the fluorescence intensity at physiological pH, electron-withdrawing groups were
CH3
CH3
1O
2 O O
CH3
CH3
9,10-Dimethylanthracene Endoperoxide
λemission = 436 nm
Fig. 10. Reaction of 9,10-dimethylanthracene with 1O2 to produce endoperoxide.

O
O
1O
2
COOH COOH
X X X X
HO O O HO O O
X = H DPAX-1 λex= 493 nm λem= 516 nm DPAX-1-EP λex= 494 nm λem= 515 nm
X = Cl DPAX-2 λex= 507 nm λem= 524 nm DPAX-2-EP λex= 506 nm λem= 527 nm
X = F DPAX-3 λex= 493 nm λem = 514 nm DPAX-3-EP λex= 494 nm λem= 515 nm
1
Fig. 11. Reaction of 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs) with O2 to
produce DPAX endoperoxides (DPAX-EPs) (adapted from Ref. [79]).
incorporated at the 2- and 7-positions of the xanthene chromophore, leading to Cl (DPAX-2) and
F (DPAX-3) (Fig. 11). This modification lowered the pKa value of the phenolic oxygen atom
[79].
DPAX-2 was used to detect the production of 1O2 from two different generation systems: the
MoO42/H2O2 system and the 3-(4-methyl-1-naphthy)propionic acid endoperoxide (EP-1)
system, which act at different pH values (10.5 and 7.4, respectively). In both cases an increase
of the probe’s fluorescence was verified when in contact with the generating system. These
results confirmed DPAXs’ advantage when detecting 1O2 in neutral or basic aqueous solutions
[79]. The behaviour of this probe towards H2O2, !NO and O2! was also studied, but no change in
the intensity of the fluorescence was observed for any of these reactive species. These facts
corroborate the specificity of this probe for 1O2 [79].
The detection of 1O2 in biological samples was also investigated. With this purpose, DPAX-2
diacetate (DPAX-2-DA) was prepared, since it was considered to be more permeable to cells.
DPAX-2-DA is hydrolysed by intracellular esterases to generate DPAX-2. Both DPAX-2 and
DPAX-2DA were tested and compared in the same assay systems. However, cells were stained
similarly in both cases. This observation probably means that DPAX-2 itself is also membrane-
permeable [79].
4.3. 9-[2-(3-Carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX)
Tanaka et al. [18] developed another fluorescent probe for detection of 1O2, the 9-[2-(3-
carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) (Fig. 12), aiming to
achieve greater sensibility and rapidity of formation of endoperoxide comparatively to the
already existent DPAX (Fig. 11). The development of this new probe was based on the
fact that DMA reacts very rapidly and specifically with 1O2 (k = 9.1 108 M 1 s 1 in
water) originating the correspondent 9,10-endoperoxide (DMA-EP) (Fig. 12). This reaction is
much more rapid than DPA’s with 1O2 (1.0 106 M 1 s 1 in benzene; no data available in
water) [18].
CH3 CH3
O
O
H3C H3C
1O
2
COOH COOH
HO O O HO O O
DMAX DMAX-EP
(Low fluorescence) (High fluorescence)
λexcitation= 492 nm
λemission= 515 nm
Fig. 12. Reaction of 9-[2-(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) with 1O2 to produce

DMAX-EP (adapted from Ref. [18]).
Similarly to DPAX, DMAX was developed through the association of fluorescein with a
fixating molecule of 1O2, DMA in this case [18,79].
DMAX and its endoperoxide (DMAX-EP) (Fig. 12) have similar excitation and emission
wavelengths (k excitation = 492 nm; k emission = 515 nm) but, while DMAX is practically non-
fluorescent, DMAX-EP presents high fluorescence. The quantum yield of DMAX-EP is about
1.5 times higher than that of DPAX-EP [18].
In the studies carried out by Tanaka et al. [18], the intensity of the fluorescence of DMAX
increased in a concentration dependent manner concerning the generator of 1O2 (EP-1), and a
good linear relation between these two parameters has been observed. This enables the use of
DMAX as a means of quantitatively detecting 1O2. Tanaka et al. [18] also confirmed the
specificity of DMAX for 1O2 when verifying that there was no change in the probe’s
fluorescence intensity in the presence of H2O2, !NO and O2!. DMAX reacts with 1O2 more
rapidly, and its sensitivity is 53-fold higher than that of DPAXs. The hydrophobicity of DMAX
is less than that of DPAXs, which is important for its use in biological samples. DMAX is,
therefore, a fluorescent probe appropriate for detecting 1O2 with a potential usefulness for assays
in biological systems [18].
5. Fluorescence probes for detection of hydroxyl radical
5.1. 4-(9-Anthroyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl
It has been shown that paramagnetic nitroxides are efficient quenchers of excited singlet
states of aromatic hydrocarbons, presumably through an intermolecular electron exchange
interaction between the ground state nitroxide and the excited state compound within a collision
complex [80]. Yang and Guo [80] synthesized a new nitroxide derivative covalently linked with
a fluorophore [4-(9-anthroyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl] (Fig. 13). In such a
hybrid molecule, both the fluorescence quenching and the radical trapping properties of the
nitroxide are advantageous in a technique for monitoring radicals. Fluorescence emission from
CH3
.
O O
H3C N CH3 H3C N CH3

H3C CH3 H3C CH3
.CH
3
O O
O C O C
(I) (II)
Low fluorescence High fluorescence

λemission = 427 nm
Fig. 13. Reaction of 4-(9-anthroyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl (compound I) with methyl radical to produce

the O-methylhydroxylamine (compound II) (adapted from Ref. [80]).
the fluorophore of this hybrid molecule is greatly quenched, presumably through an electron
exchange mechanism, which both reduces the fluorescence quantum yield and shortens the
fluorescence lifetime. However, the reaction of the hybrid molecule with a carbon-centred
radical, or chemical reduction to its corresponding hydroxylamine, leads to the formation of a
diamagnetic product, thereby eliminating the intramolecular quenching pathway and resulting in
a large enhancement in fluorescence emission [80–82]. Indeed, the synthesized anthracene–
nitroxide hybrid molecule (compound I) showed an indirect sensitive response to HO!. The
response was based on the reaction of HO! with DMSO to produce quantitatively a methyl
radical (!CH3) (Eq. (2)), which then combined with I to produce a stable O-methylhydrox-
ylamine (compound II) resulting in a large enhancement in fluorescence intensity
(k excitation = 377 nm; k emission = 427 nm) (Fig. 13) [80]. The fluorescence increase was
proportional to the amount of !CH3 generated from the reaction of DMSO with HO!, which,
in turn, was proportional to the concentration of HO!.
HO! þ ðCH3 Þ2 SO Y CH3 SO2 H þ ! CH3 ð2Þ
This methodology is simple, specific, and easy to operate and has a relatively high sensitivity.
Unlike the aromatic hydroxylation method, only one quantitative product (compound II) is
produced in the detection system, thus making the quantitative analysis simple. The quantitative
reaction of HO! with DMSO can also be obtained at a relatively high DMSO concentration with
little adverse effect on the fluorescence signal [80]. However, the methodology also suffers from
some limitations. One is that the nitroxide moiety of the hybrid molecule (compound I) can be
metabolised to its corresponding hydroxylamine in the presence of cellular reductants (e.g.
ascorbic acid, GSH, NADPH), and hence cause an overestimate of HO! production [80].
Another drawback is that other carbon-centred radicals will also couple with the hybrid molecule
(I) and hence give rise to a fluorescence increase in the detection system. This problem can be
prevented if the present method is coupled to an HPLC with post-column detection of the O-
methylhydroxylamine derivative (compound II) [80].
5.2. 1,3-Cyclohexanedione (CHD)
1,3-Cyclohexanedione (CHD) is a non-fluorescent compound that can be used for the indirect
measurement of HO!. This methodology makes use of the reaction between HO! and DMSO,
which generates formaldehyde (Eqs. (3), (4), and (5)). Formaldehyde then reacts with ammonia
and CHD at pH 4.5 to generate a new compound that, when heated at 95 8C for 20 min or kept at
room temperature overnight, becomes fluorescent (k excitation = 400 nm; k emission = 452 nm) (Fig.
14) [83]. Although the proposed method for the detection of HO! was considered simple,
sensitive and easy to operate, the indirect measurement by the authors, with four reactions
needed to generate a compound that needs to be heated at 95 8C for 20 min, may lead to great
difficulties in interpreting the obtained data.
HO! þ ðCH3 Þ2 SO Y CH3 SO2 H þ ! CH3 ð3Þ
!
CH3 þ O2 Y CH3 OO! ð4Þ
2CH3 OO! Y HCHO þ CH3 OH þ O2 ð5Þ
5.3. Sodium terephthalate
Sodium terephthalate is a non-fluorescent compound that reacts with HO! and originates
an aromatic hydroxylated product, sodium 2-hydroxyterephthalate, which has strong fluo-
rescence (k excitation6310 nm; k emission6430 nm) (Fig. 15) [84]. Accordingly, Tang et al.
[84], developed a method using sodium terephthalate as the trapper of HO!, using H2O2
and Co2+ as the HO! source and a flow injection spectrofluorimeter to measure the fluo-
rescence product. The optimum pH range for the aromatic hydroxylation was 6.80–7.90. The
proposed method was applied to verify the scavenging effect of thiourea and mannitol in
which there was a quantitative correlation between the concentration and the scavenging
effect.
O O
O O
HCHO
NH3 N
1,3-Cyclohexanedione H
λemission = 452 nm
Fig. 14. Reaction of 1,3-cyclohexanedione with formaldehyde in the presence of ammonia (adapted from Ref. [83]).
COO- COO- C O
H
OH
HO. O
COO- COO- COO-

Terephthalate 2-Hydroxyterephthalate
Fig. 15. Reaction of terephthalate with HO! to produce 2-hydroxyterephthalate (adapted from Ref. [36]).
5.4. Coumarin, coumarin-3-carboxylic acid (3-CCA) and N-succinimidyl ester of

coumarin-3-carboxylic acid (SECCA)
Coumarin and its non-hydroxylated derivatives are non-fluorescent under physiological

conditions. However, hydroxylation of these compounds by HO! generates fluorescent products
[85–87]. The fluorescence of hydroxylated coumarins strongly depends on the site of
hydroxylation of the aromatic ring. Thus, for accurate quantification of HO!, it is essential to
identify the coumarin hydroxylation products. The positions of hydroxylation depend on the
conditions of HO! generation and may also depend on the structure of the coumarin derivative
[85,88]. The major known product of hydroxylation of coumarin is 7-hydroxycoumarin (7-OHC,
umbelliferone). It has high quantum yield (~0.5, at normal physiological conditions), although it
depends on pH [85,88].
The substitution of the hydrogen in C-3 with the carboxylic group of coumarin resulted in
coumarin-3-carboxylic acid (3-CCA) (Fig. 16). The use of 3-CCA as a probe for HO! was shown
to be advantageous, comparatively to coumarin: the carboxylic group in C-3 eliminates
hydroxylation in this position and increases the fluorescence of 7-hydroxylated coumarin
derivatives two fold [89,90]. Indeed, the hydroxylation of 3-CCA by HO! generated by radiation
or by chemical means produces 7-HOCCA, which is easily detectable by fluorescence. The
emission and excitation spectrum of 7-HOCCA has an emission maximum of 450 nm and
excitation bands at 350 and 395 nm [88,91]. Quantification of HO! using 3-CCA was
demonstrated to be accurate, reproducible and performed in real time. However, like other
hydroxylated coumarins, 7-HOCCA is highly sensitive to pH, which requires careful control of
pH during detection [88].
3-CCA has been successfully used, in vitro, for the screening of HO! scavenging activity [92]
and HO! generation activity [93] of several compounds.
O O
COOH C O N
O
O O O O
3-CCA SECCA
Fig. 16. Chemical structure of coumarin-3-carboxylic acid (3-CCA) and N-succinimidyl ester of coumarin-3-carboxylic
acid (SECCA).
3-CCA has also been derivatized to its succinimidyl ester (SECCA) (Fig. 16) and coupled to
free primary amines of albumin, avidrin, histone-H1, polylysine, and an oligonucleotide. The
rationale for the use of these derivatives is that SECCA is bound to the biomolecule being
irradiated, therefore the induced fluorescence is a measure of the presence of HO! in the vicinity
of the biomolecule and hence should be related to the concomitant HO! attack. When SECCA-
biomolecule conjugates are exposed to HO!, generated by radiation or chemically, the
relationship between induced fluorescence and HO! concentration was linear in the examined
concentration range. The fluorescence excitation spectrum of irradiated SECCA-biomolecule
conjugates was very similar to that of 7-HO–SECCA-biomolecule conjugates, indicating the
conversion of SECCA to 7-HO–SECCA following irradiation [91,94–99].
Control studies in environments that excluded certain radiation-induced water radicals for
both the conjugated and unconjugated forms of irradiated SECCA demonstrated that for free
SECCA the induction of fluorescence: (i) shows a linear dependence on radiation dose; (ii) is
mediated specifically by the HO!; (iii) is not significantly affected by other water radicals, and
(iv) is enhanced by a factor of 1–4 in the presence of oxygen. For SECCA-biomolecule
conjugates it was similarly shown that the induction of fluorescence on SECCA-histone-H 1 or
SECCA-oligonucleotide: (a) is linearly related to radiation dose and is mediated by the HO!, in
the absence of which no fluorescence is induced, (b) is not significantly affected by primary
water radicals other than HO! or by higher order radicals formed on the biomolecules examined,
and (c) is enhanced in the presence of oxygen by a factor of about 1–4 [91].
5.5. 2-[6-(4V-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and

2-[6-(4V-amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF)
2-[6-(4V-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2-[6-(4V-amino)-

phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF) are non-fluorescent derivatives of
fluorescein, designed and synthesized by Setsukinai et al. [100] that originate fluorescein by
O-dearylation, leading to its characteristic fluorescence (k excitation = 500 nm; k emission = 520 nm)
(Fig. 17).
Setsukinai et al. [100] studied the reactivity of these two probes for several ROS and RNS.
Both probes revealed to be capable of detecting HO! generated by a Fenton reaction, originating
a fluorescence increase in a concentration dependent manner. This fluorescence increase was
suppressed in the presence of DMSO, a scavenger of HO!. APF revealed to be reactive for
HOCl, contrarily to HPF. The fluorescence of both probes also increased in the presence of
ONOO. However the reactive species H2O2, !NO, O2!, 1O2 and ROO! did not cause any
modification in the fluorescence of the probes.
These probes were also studied in the presence of an enzymatic system, being verified that an
HRP/H2O2 system causes a fluorescence increase in both HPF and APF in a concentration
dependent manner. The MPO/H2O2/Cl system causes a fluorescence increase in APF in a
concentration dependent manner but it does not cause any fluorescence increase in HPF. This
result was to be expected regarding that only APF reacts with HOCl [100].
APF and HPF were classified by Setsukinai et al. [100] as appropriate probes for detecting
highly reactive ROS and RNS, meaning, HO!, ONOO and HOCl. According to these authors,
this constitutes an advantage of these probes relatively to DCFH in what concerns selectivity,
being that this last demonstrated to be reactive for all ROS and RNS, in an experiment carried
out by the same authors. Besides, APF and HPF present high resistance to auto-oxidation,
contrarily to DCFH and can be used in enzymatic and cellular systems. Thus, although the
XH
O O O O O O-
COO- COO-
X O
X=O HPF (Strongly fluorescent)

X = NH APF
(Almost non fluorescent) λexcitation = 500 nm
λemission = 520 nm
Fig. 17. Reaction scheme of 2-[6-(4V-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2-[6-(4V-

amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF) O-dearylation induced by reactive species (adapted from
Ref. [100]).
sensibility of DCFH is higher than that of APF and of HPF, the use of these last when detecting
certain ROS and RNS might be worthwhile.
5.6. Fluorescein (FL)
Fluorescein (FL) is a well-known fluorescent compound (k excitation = 495 nm, k emission = 515
nm) that is prone to be oxidized by several reactive species into a non-fluorescent product,
namely by HO!. This reactivity has been shown to be useful for the assessment of antioxidant
activity in an assay known as bHydroxyl radical averting capacity (HORAC)Q [101]. In this
assay, the generation of HO! induced by metal ions (M) is monitored by the fluorescence
decrease of FL (Eq. (6)). The putative antioxidant may prevent FL oxidation by inhibiting HO!
formation, due to inactivation of metal ions reactivity, or by reacting itself with formed HO!.
FL þ MðIIÞ þ H2 O2 Y oxidized FLðloss of fluorescenceÞ ð6Þ
Consequently, the inhibition degree on Eq. (6) is the antioxidant capacity index, which can be
quantified through fluorescence curves [101].
6. Fluorescence probes for detection of peroxyl radical
6.1. cis-Parinaric acid (cis-PnA, (18:14):9,11,13,15-cis-trans-trans-cis-octadecaenoic acid)
cis-Parinaric acid (cis-PnA, (18:14):9,11,13,15-cis-trans-trans-cis-octadecaenoic acid) (Fig.

18) is a fluorescent 18-carbon polyunsaturated fatty acid with a linear polyenic structure,
consisting of 4 conjugated p-electron bonds (k excitation = 320 nm; k emission = 432 nm). Taking into
account that the fluorescence of cis-PnA is irreversibly lost upon oxidation, this compound has
been used as a probe to evaluate lipid peroxidation [102,103] and/or for the evaluation of the
antioxidant activity of lipophilic compounds [104].
COOH
cis-Parinaric acid
λemission = 432 nm
Fig. 18. Chemical structure of cis-parinaric acid.
To Drummen et al. [103], the use of cis-PnA as a membrane probe is suitable,

straightforward, sensitive and reproducible for detecting the initial stages of lipid peroxidation
in living cells, with several other advantages: (i) it is in a conformation closely resembling the
endogenous fatty acyl moieties, with a mobility that is most likely comparable to endogenous
phospholipids; (ii) the fluorescent and peroxidative properties are combined in the conjugated
system of unsaturated carbon–carbon bonds; (iii) its fluorescence properties are instantaneously
lost upon peroxidation; (iv) it does not contain a bulky reporter group and as such does not
perturb the lipid bilayer, and (v) it can be metabolically integrated into membrane phospholipids
of cultured cells. Thus, this probe has been extensively used to measure lipid peroxidation in
lipoproteins, erythrocytes, sub-mitochondrial particles, sarcoplasmic reticulum, rat aortic smooth
muscle cells, lens membrane, rapid dividing cell lines, and macrophages (in Ref. [103]).
Nonetheless, there are some problems associated with the use of cis-PnA for measuring the
activity of lipophilic antioxidants in lipid environments. In principle, the cis-PnA assay is an
endpoint assay, requiring extraction of probe and lipids, thus decreasing its value for direct
measurement of fluorescence in living cells [105]. cis-PnA absorbs in the UV region at 320 nm,
where most test compounds also absorb. cis-PnA is also especially air sensitive and photolabile
and undergoes photodimerization under illumination, which results in loss of fluorescence [104],
and leads to an overestimation of the extent of lipid peroxidation. Thus, experimental results
should always be precautiously interpreted.
6.2. 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic
acid (C11-BODIPY581/591)
4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid
(C11-BODIPY581/591) is a fluorescent probe (k excitation = 510 nm; k emission = 595 nm) (Fig. 19)
used for evaluating lipid peroxidation and antioxidant efficacy in model membranes,
lipoproteins, biological fluid and living cells.
C11-BODIPY581/591 has a long chain fatty acid (C11) as a substitute. The non-polar character
of C11 makes C11-BODIPY581/591 liposoluble (the octanol/water partition coefficient for C11-
BODIPY581/591 is 4.57) and its number of double conjugated links makes it susceptible to
oxidation by ROO! [104,106]. The rate constant for the reaction of C11-BODIPY581/591 with
ROO! was estimated as 6.0 103 M 1 s 1, which is about two orders of magnitude larger than
the rate constant for the reaction of ROO! with polyunsaturated lipids [106]. On the other hand,
the reaction of C11-BODIPY581/591 with ROO! is almost completely inhibited by a-tocopherol, a
potent scavenger of this radical [106]. This makes C11-BODIPY581/591 kinetically an inefficient
probe especially in the presence of potent radical-scavenging antioxidants such as tocopherols,
but a convenient probe for lipid peroxidation. Thus, C11-BODIPY581/591 may be used as an
efficient probe for the free radical-mediated oxidation that takes place in the lipophilic domain,
+
N N
B
(CH=CH)2 F F (CH2)10-COOH
C11-BODIPY581/591
λemission = 595 nm
Fig. 19. Chemical structure of C11-BODIPY581/591 (adapted from Ref. [104]).
especially after depletion of a-tocopherol, while it may not be an efficient probe for detection of
aqueous radicals. Indeed this probe has excellent characteristics for measuring ROO!-mediated
lipid peroxidation: (i) it has fluorescent properties in the red visible zone of the spectre
(k emission = 595 nm), which allows its use in fluorescence microscopy and its application in
fluorescence microplate readers; (ii) during oxidation induced by reactive species its
fluorescence turns to green (k emission = 520 nm). This characteristic is highly advantageous
because it allows the observation of oxidizing activities at subcellular levels; (iii) has a high
quantum yield and because of this, low labelling concentrations can be used, ensuring minimal
perturbation of the membrane whilst retaining favourable signal to noise ratios; (iv) has a good
photo-stability and displays very few fluorescence artefacts; (v) is virtually insensitive to
environmental changes, i.e., pH or solvent polarity; (vi) is lipophilic and as such easily enters
membranes; (vii) once oxidized, C11-BODIPY581/591 remains lipophilic and does not
spontaneously leave the lipid bilayer; (viii) C11-BODIPY581/591 localizes in two distinct pools
within the lipid bilayer, a shallow pool at 18 Å and a deep pool at b7.5 Å from the centre of the
bilayer; (ix) its low cytotoxicity in low labelling concentration ensures minimal perturbation of
cellular processes, whilst still retaining favourable signal-to-noise ratios; (x) its sensitivity to
oxidation is comparable to that of endogenous fatty acyl moieties [104,107,108].
Direct measurement of fluorescence in living cells may constitute a major advantage
compared with the cis-PnA assay, which in principle is an endpoint assay, requiring extraction of
probe and lipids [105].
Naguib [104] applied this probe when detecting ROO!, using 2,2V-azobis-2,4-dimethylvaler-
onitrile (AMVN) as generator of ROO!. This author suggested that this is a valid way of
monitoring lipophilic antioxidants contrarily to the methodology of Cao et al. [109] where an
aqueous medium and the non-lipophilic generator of ROO! 2,2V-azobis(2-amidinopropane)
dihydrochloride (AAPH) are used, which can present a reactivity towards lipophilic antioxidants
different from the one of those generated by AMVN [104].
C11-BODIPY581/591 is sensible to multiple oxidizing species. It was demonstrated that this
probe is oxidized by ROO!, HO!, RO!, ONOO being, nevertheless, insensible to H2O2, 1O2,
O2!, to !NO, to transition metals and to hydroperoxides, when these are not in the presence of
transition metals [106,107]. The primary target for these reactive species in the C11-
BODIPY581/591 molecule is the diene interconnection, leading to the formation of three
different oxidation products that are responsible for the observed shift from red to green
fluorescence [108]. Two of these products are closely related and are generated after oxidation
by a wide variety of radicals. The third oxidation product, corresponding to a hydroxylated
variety of the intact probe, was the only oxidation product observed after exposure of
C11-BODIPY581/591 in homogeneous ethanolic solution to ROO!, but was recovered only in

small amounts from cells that were exposed to this radical [108].
This probe presents, however, an unfavourable characteristic that is the fact of degrading
itself under high intensity illumination conditions, which are unleashed in laser confocal
microscopy [107].
6.3. Lipophilic fluorescein derivatives
Makrigiorgos et al. [110] used 5-(N-dodecanoyl)aminofluorescein (C11-fluor) (Fig. 20), a

lipophilic derivative of fluorescein, for determining the lipid peroxidation associated to the
cellular membrane, by flow cytometry. This probe’s capacity to remain connected to the cellular
membrane in a stable and irreversible way keeps it close to the generation site of ROO!, which
makes C11-fluor a more sensible probe comparatively to others that are used in solution such as
5- and 6-carboxyfluorescein [110].
In the study of Makrigiorgos et al. [110] the fluorescence of C11-fluor, connected to the
cellular membrane of red blood cells, was gradually quenched in the presence of cumene
hydroperoxide, an initiator of lipid peroxidation in this type of cells. This fluorescence
quenching became slower in presence of Trolox and vitamin E, well-known scavengers of
ROO!. The same was verified when using the already established cis-PnA method, which served
HO O O
COOH
N CO (CH2)nCH3
H
n = 10 (C11-fluor)
n = 14 (C16-fluor)
n = 16 (C18-fluor)
HO O O
O
COOH
H2CO C (CH2)14CH3
O HCO C (CH2)14CH3
N C NHCH2CH2O P OCH2 O
H
S O
(fluor-DHPE)
Fig. 20. Chemical structure of lipophilic derivatives of fluorescein: 5-(N-dodecanoyl)aminofluorescein (C11-fluor), 5-

hexadecanoylaminofluorescein (C16-fluor), 5-octadecanoyl-aminofluorescein (C18-fluor) and dihexadecanoylglyceropho-
spho-ethanolamine (fluor-DHPE).
as comparison in this study. These results, associated to the excitation wavelength of fluorescein
(488 nm), favourable to the use of laser sources and cells, make this probe appropriate to be used
in flow cytometry and in fluorescence microscopy [110].
Furthermore, Maulik et al. [111] studied four lipophilic derivatives of fluorescein C11-fluor, 5-
hexadecanoylaminofluorescein (C16-fluor), 5-octadecanoyl-aminofluorescein (C18-fluor) and
dihexadecanoylglycerophosphoethanolamine (fluor-DHPE) (Fig. 20) in what concerns their
capacity of exchanging between labelled and unlabeled cells, having verified that only fluor-
DHPE does not exchange between cells. Thus, this probe is, amongst all four, the only one that
can identify the differences that might exist between cellular subpopulation in what concerns
lipid peroxidation [111].
Maulik et al. [111] reported that the type of cells (erythrocytes) used in their study has
characteristics that facilitate lipid peroxidation. However, it will be necessary to confirm if fluor-
DHPE is a probe sensible enough to be used with other types of cells.
6.4. Dipyridamole
Dipyridamole is a fluorescent compound (k excitation = 415 nm; k emission = 480 nm) (Fig. 21).
The tertiary nitrogens in the aliphatic chains and in the aromatic pyrimido-pyrimidine nucleus
are responsible for an intense absorption band in the 400–480 nm regions and for an intense
fluorescence [112,113].
This compound is a well-known pharmaceutical drug used in medicine as a coronary
vasodilator and anti-platelet agent. It was previously shown that dipyridamole is also a potent
antioxidant that reacts with HO!, O2!, and ROO! [114–116]. Taking into account that this
reactivity leads to fluorescence quenching, Iuliano et al. [113] suggested the use of dipyridamole
and some of its derivatives as fluorescent probes for these radicals in biological systems. These
authors demonstrated that the fluorescence of dipyridamole lowers gradually in time when it is
oxidized by ROO! generated by the thermal dissociation of azo-initiator compounds (namely by
AAPH). In its turn, the oxidation of dipyridamole is inhibited by certain antioxidants such as a-
tocopherol. Iuliano et al. [113] also tested five dypirydamole derivatives, which showed
comparable rate constants towards ROO!.
OH
N
N N
HO N
N OH
N N
N
HO
Dipyridamole
λemission = 480 nm
Fig. 21. Chemical structure of dipyridamole (adapted from Ref. [113]).

The rate of dipyridamol fluorescence decay induced by AAPH is largely affected by pH. The
rate of decay is very slow in the pH range 5–6, exhibits a rapid increase as the pH is raised from
6 to 7 units and plateaus in the pH range 7–8. This effect is dependent on dipyridamole since its
fluorescence is sensitive to pH [113].
The advantage of using dipyridamole as a probe is its chemical stability. Indeed, dipyridamole
is not air-sensitive and is stable. Ethanolic solutions of dipyridamole are stable at room
temperature over months [113]. Nevertheless, it must be stored in the dark since dipyridamole is
not photostable under aerobic conditions [117]. Dipyridamole is soluble in water and in organic
solvents, which enables the assessment of the activity of hydrosoluble or liposoluble
antioxidants.
6.5. Diphenyl-1-pyrenylphosphine (DPPP)
Diphenyl-1-pyrenylphosphine (DPPP) is a non-fluorescent compound that is known to react

stoichiometrically with hydroperoxides to give diphenyl-1-pyrenylphosphine oxide (DPPPO), a
fluorescent product (k excitation = 351 nm; k emission = 380 nm), and alcohol (Fig. 22) [118]. Using
this rationale, plasma levels of hydroperoxides of phosphatidylcholine, phosphatidylethanol-
amine, triglycerol and cholesteryl esters have been determined by HPLC post-column detection
systems by using DPPP [119,120]. Because of its high reactivity against hydroperoxides as a
reductant and because of especially high yield of fluorescence of the resulting product, DPPP has
proved to be a sensitive probe for lipid hydroperoxides. Thus, DPPP was subsequently used as a
fluorescent probe for the measurement of the extent of oxidation in solution and in low-density
lipoprotein particles [121,122] and to monitor lipid peroxidation in living cell membranes
[123,124].
After incubating mouse polymorphonuclear leukocytes (PMNs) with DPPP as a DMSO
solution, Okimoto et al. observed that DPPP diffused into cells and was retained in the cell
membranes [123]. Using DPPP, these authors showed that, similar to the liposomal system,
DPPP in cells showed remarkably high reactivity toward MeLOOH compared with H2O2 and
that lipid peroxidation proceeded in PMNs upon stimulation with phorbol 12-myristate 13-
P: ROOH P O + ROH
DPPP DPPP=O
λemission = 380 nm
Fig. 22. Reaction of diphenyl-1-pyrenylphosphine (DPPP) with hydroperoxide to produce diphenyl-1-pyrenylphosphine

oxide (DPPPO) and alcohol (adapted from Ref. [123]).
acetate (PMA). Okimoto et al. [123] concluded that this novel method with DPPP has several
advantages: (i) DPPP reacts with peroxides stoichiometrically, that is, in mol–mol ratio, by a
straightforward mechanism to give DPPPO, which shows that this is suitable for a sensitive
and quantitative measurement of lipid hydroperoxides; (ii) DPPP is highly lipophilic and
reacts with lipid hydroperoxides selectively and does not react with aqueous peroxides. This is
in contrast to well-known fluorescent probes such as DCFH and DHR. It also implies that
only the lipid peroxidation taking place in the membranes can be selectively measured with
DPPP; (iii) the use of DPPP enables the continuous observation of oxidation in cells and
tissues in a non-destructive manner.
Success of DPPP as a probe for long-term peroxidation in live cells may lie in the
biologically inactive structure of this molecule. Their artificial, non-biological structures
probably make these molecules resistant to catabolic and metabolic activities of the cell. As a
probe to be used in live cells for a relatively long period of time this property should be quite
important [124].
The disadvantage of this method may be that the fluorescence emission wavelength of
DPPPO (380 nm) is near the ultraviolet region.
6.6. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA)
As mentioned above, 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) has been used for

detecting several ROS and RNS in biological media. Cellular esterases first hydrolyse DCFH-
DA to DCFH, which is then oxidized by reactive species and originates 2,7-dichlorofluorescein
(DCF), a fluorescent compound (k excitation = 498 nm; k emission = 522 nm). DCFH is soluble in
lipids and reacts with radicals in the lipophilic compartment as well as in the aqueous phase (the
octanol/water partition coefficient for DCFH is 2.62). The reaction of DCFH with aqueous
radicals is suppressed by aqueous antioxidants, whereas that with lipophilic radicals is not [105].
The utility of this probe for detecting lipid hydroperoxides has long been referred by Cathcart et
al. [125].
The ability for an antioxidant compound to scavenge ROO! is usually referred as bTotal
peroxyl radical trapping potentialQ (TRAP), which has been used to compare the in vitro
antioxidant potency of several compounds [126]. The assessment of TRAP has also been
used as a way of measuring the antioxidant activity of serum or plasma [127]. The
measurement of TRAP in the serum is based on the amount of time that serum is capable
of resisting to artificially induced peroxidation [126]. This measurement can be performed
through different methodologies [127]. A fluorescence methodology to determine TRAP
was suggested by Valkonen and Kuusi [126] using AAPH to generate ROO! and DCFH-
DA as the oxidizable substrate for the ROO!. Serum esterases are important for the
hydrolysis of DCFH-DA into DCFH, whose oxidation by ROO! leads then to the formation
of DCF.
In the TRAP assay, DCF fluorescence or absorbance formation contains four phases. The first
lag phase is due to the antioxidants in the sample. After the consumption of antioxidants by
ROO!, the reaction proceeds to the first propagation phase, which is due to the fluorescence
increase during the reaction of DCFH-DA with ROO!. The second lag phase (T Trolox), which
interrupts the first propagation, is due to the action of Trolox, a liposoluble analogous to vitamin
E (the internal standard), added during the first propagation phase. The second propagation stage
starts after the consumption of Trolox. The first lag phase is compared to the second lag phase
and, thus, related to the antioxidant capacity of the sample [127].
The calculation of the TRAP value is represented by the following equation (adapted from
Ref. [126]):
TRAP ¼ ðTserum = TTrolox Þ serum dilution factor 2 ½Trolox ð7Þ
The antioxidant supplements introduced in the diet and some physiologic and patophysio-
logic states have been related with altering in the TRAP values, although the relevance of these
relations is yet to be clarified [126].
6.7. b-Phycoerythrin/Fluorescein/6-Carboxyfluorescein
The ability for scavenging oxygen radicals, namely ROO!, was developed by Cao et al. [109]
with the aim of assessing the antioxidant activity of human serum and has been referred as
Oxygen Radical Absorbance Capacity (ORAC) assay.
ORAC is a fluorescence technique that requires the use of a fluorescent indicator whose
fluorescence decreases due to its oxidation by reactive species (ROO! is the reactive species that
is being used) [109,128,129]. Cao et al. [109] used h-phycoerythrin as indicator, an
hydrosoluble protein isolated from Porphyridium cruentum, which absorbs visible light,
presenting high fluorescence yield and sensibility to ROS [128]. This protein has, however,
some disadvantages such as its inconsistency from lot to lot, which results in a variable reactivity
with ROO!, its instability before light that might lead to fluorescence loss when the probe is
exposed to the excitation wavelength for a certain amount of time, a situation that was observed
when trying to adapt this methodology to a plate reader; its interaction with polyphenols, the
major category of antioxidants present in natural products, due to non-specific protein links.
Because of this there have been used other fluorescent indicators such as fluorescein [130] and
one of its derivatives, 6-carboxyfluorescein [128]. 6-Carboxyfluorescein has characteristics such
as its high quantum yield, its high molar absorptivity and its high thermo and photochemical
stability that make it a good fluorescent indicator [130]. Fluorescein also presents a high
quantum yield at pH N 7, is not expensive, is stable to light and does not present interaction with
other compounds. The restriction of fluorescein’s pH does not weight much in this methodology
for the samples are quite diluted in buffer (pH 7.4) due to its high sensibility [128].
AAPH is used in this technique as a generator of ROO!, which reacts with the fluorescent
indicator, causing a decrease in its fluorescence at a rate that depends on the production rate of
ROO! [109,130].
The ORAC test depends on the oxidation degree of a fluorescent probe by ROO!,
demonstrated by the intensity change in its fluorescence. The inhibition of this oxidation by an
antioxidant is, in the ORAC test, a measurement of the antioxidant activity of that substance for
the free radical. The peculiarity of the ORAC test is that the reaction between the probe and the
free radical is measured as a time course effect, being the quantification of the inhibition
obtained by calculating the area under curve (AUC). This calculation enables to combine time
with the inhibition degree of the free radicals by the antioxidant in one single value [131].
The ORAC (Eq. (8)) value is expressed relatively to an antioxidant used as calibration
standard. Trolox is the one that is commonly used in this technique [128].
Relative ORAC value ¼ ½ðAUCSample AUCBlank Þ = ðAUCTrolox AUCBlank Þ

ðmolarity of Trolox = molarity of sampleÞ ð8Þ
The ORAC methodology is useful not only in serum samples, but also in samples containing
antioxidants whose ORAC value aims to be determined. Cao et al. [109] found a linear relation
between the ORAC value and the concentration of antioxidants present in the samples. This test
was successfully applied in the assessment of a-tocopherol, h-carotene and bilirubin, suggesting
that AAPH, although hydrosoluble, is capable of reacting with liposoluble antioxidants [109].
On the other hand, this methodology was used by Naguib [130] only to determine the activity of
hydrosoluble antioxidants. In fact, one of the disadvantages that has been pointed out in the
methodology initially developed by Cao et al. [109] is that it does not allow the determination of
the ORAC value of lipophilic compounds since the test is developed in aqueous solution
[129,132]. To overcome this obstacle, there have been used randomly methylated h-
cyclodextrins (RMCD) to promote the solubility of the lipophilic antioxidants in aqueous
solution. RMCD do not interfere with the methodology because they do not have antioxidant
activity nor do they prevent an antioxidant to chemically function as such [129]. Prior et al.
[132], in turn, suggest a methodology where it is possible to determine the ORAC of the
lipophilic and hydrophilic stages on the same sample, which enables to obtain, by summing both
values, the sample’s total antioxidant activity.
Huang et al. [131] adapted this methodology to a plate reader with an automatic sampling
system, using fluorescein as fluorescent probe. This system allows to substantially shorten the
time of the test and to eliminate human errors in the different steps for preparation of the samples
[131].
7. Final comments
As mentioned above, reactive species present some characteristics that make them difficult to
detect, namely their very short lifetime and the variety of antioxidants existing in vivo, capable
of capturing these reactive species. Although the market has already several offers concerning
fluorescent probes, the choice of the most adequate should be criteriously made in order to
assure the best result in the experimental conditions under study. Several factors concerning the
fluorescence probes must be taken into account, namely (i) the relative solubility in aqueous and
lipid environments; (ii) the ability to cross cellular membranes and intracellular distribution; (iii)
the specificity and sensitivity of the reaction with the ROS intended to be measured; (iv) the
possible need of enzymatic systems; (v) the required incubation time; (vi) the required sample
handling; (vii) the interference from the pH, solvents, or other biological and non-biological
experimental conditions in the system under study; (viii) the chemical-, thermal- and photo-
stability of the probe and/or fluorescent product; (ix) the excitation and emission wavelength of
the fluorescent product.
The fluorescence probe must be competent to be used to quantify the reactive species
generated under non-biological and biologically relevant conditions. Signal transduction
hampering due to the scavenging of the reactive species by the probes and the possible
toxicity of probes and/or UVexcitation light should also be factors to be taken into account.
Hopefully, the present review will help readers to find the probe that better fits their scientific
objectives.
Acknowledgments
The authors greatly acknowledge FCT and FEDER financial support for the project POCTI/
QUI/59284/2004.
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Fluorescence Probes Used For Detection of Reactive Oxygen Species

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Fluorescence probes used for detection of reactive

Abbreviations: ROS, reactive oxygen species; O! ! !

6.5. Diphenyl-1-pyrenylphosphine (DPPP) . . . . . . . . . . . . . . . . . . . . . . . . . 71

2. Fluorescence probes for detection of superoxide radical

2.1. Hydroethidine (dihydroethidium; HE)

H2N NH2 H2N NH2

Fig. 1. Chemical structure of hydroethidine (HE) and ethidium (E+).

2.2. 1,3-Diphenylisobenzofuran (DPBF)

1,3-Diphenylisobenzofuran (DPBF) is a molecule which, under certain conditions, presents

Fig. 2. Chemical structure of 1,3-diphenylisobenzofuran (DPBF).

3. Fluorescence probes for detection of hydrogen peroxide

3.1. 2,7-Dichlorodihydrofluorescein (DCFH)

The oxidation of 2,7-dichlorodihydrofluorescein (DCFH) originates 2,7-dichlorofluorescein

Fig. 3. Oxidation of 2-(2-pyridil)-benzothiazoline by O!

Fig. 4. Mechanism of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) de-esterification to 2,7-dichlorodihydro-

Fig. 5. Mechanism of 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) de-esterification to 2,7-dichlorodihydro-

3.2. Scopoletin (7-hydroxy-6-methoxy-coumarin)

Scopoletin (7-hydroxy-6-methoxy-coumarin) (Fig. 6) is a naturally occurring fluorescent

Fig. 6. Chemical structure of scopoletin.

3.3. N-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red)

N-Acetyl-3,7-dihydroxyphenoxazine (Amplex Red) is a non-fluorescent molecule that when

Amplex Red Resorufin Resazurin

3.4. Homovanillic acid (4-hydroxy-3-methoxy-phenylacetic acid; HVA)

Homovanillic acid (4-hydroxy-3-methoxy-phenylacetic acid; HVA) represents an important

Homovanillic acid Fluorescent dimer

methodology allows the elimination of interference caused by the presence of mitochondria

3.5. Dihydrorhodamine 123 (DHR)

Dihydrorhodamine 123 (DHR) is a non-fluorescent molecule that, by oxidation, yields

Dihydrorhodamine 123 Rhodamine 123

is not directly oxidizable by H2O2 alone, by O2!, and by xanthine/xanthine oxidase

4. Fluorescence probes for detection of singlet oxygen

4.1. 9,10-Dimethylanthracene (DMA)

9,10-Dimethylanthracene (DMA) is a fluorescent compound (k excitation = 375 nm, k emission =

4.2. 9-[2-(3-Carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs)

Fig. 10. Reaction of 9,10-dimethylanthracene with 1O2 to produce endoperoxide.

4.3. 9-[2-(3-Carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX)

Fig. 12. Reaction of 9-[2-(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) with 1O2 to produce

5. Fluorescence probes for detection of hydroxyl radical

H3C N CH3 H3C N CH3

Low fluorescence High fluorescence

Fig. 13. Reaction of 4-(9-anthroyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl (compound I) with methyl radical to produce

5.2. 1,3-Cyclohexanedione (CHD)

2CH3 OO! Y HCHO þ CH3 OH þ O2 ð5Þ

5.3. Sodium terephthalate

COO- COO- COO-

5.4. Coumarin, coumarin-3-carboxylic acid (3-CCA) and N-succinimidyl ester of

Coumarin and its non-hydroxylated derivatives are non-fluorescent under physiological

5.5. 2-[6-(4V-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and

2-[6-(4V-Hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2-[6-(4V-amino)-

X=O HPF (Strongly fluorescent)

Fig. 17. Reaction scheme of 2-[6-(4V-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2-[6-(4V-

5.6. Fluorescein (FL)

6. Fluorescence probes for detection of peroxyl radical

6.1. cis-Parinaric acid (cis-PnA, (18:14):9,11,13,15-cis-trans-trans-cis-octadecaenoic acid)

cis-Parinaric acid (cis-PnA, (18:14):9,11,13,15-cis-trans-trans-cis-octadecaenoic acid) (Fig.

Fig. 18. Chemical structure of cis-parinaric acid.

To Drummen et al. [103], the use of cis-PnA as a membrane probe is suitable,

Fig. 19. Chemical structure of C11-BODIPY581/591 (adapted from Ref. [104]).

C11-BODIPY581/591 in homogeneous ethanolic solution to ROO!, but was recovered only in

6.3. Lipophilic fluorescein derivatives

Makrigiorgos et al. [110] used 5-(N-dodecanoyl)aminofluorescein (C11-fluor) (Fig. 20), a

Relative ORAC value ¼ ½ðAUCSample AUCBlank Þ = ðAUCTrolox AUCBlank Þ