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 *, Jose L.F.C. Lima

REQUIMTE, Departamento de Quımica-Fısica, Faculdade de Farmacia, Universidade do Porto, Rua Anıbal 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

0165-022X/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.jbbm.2005.10.003

Abbreviations: ROS, reactive oxygen species; O2!�, superoxide radical; HO2

!, hydroperoxyl radical; HO!, hydroxy

radical; ROO!, peroxyl radical; RO!, alkoxyl radical; H2O2, hydrogen peroxide; 1O2, singlet oxygen; HOCl

hypochlorous acid; UV, ultraviolet; RNS, reactive nitrogen species; !NO, nitric oxide; !NO2, nitrogen dioxide radical

ONOO�, peroxynitrite anion; ONOOH, peroxynitrous acid; ONOOCO2�, 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; Km

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-xanthen3-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-2,4dimethylvaleronitrile; 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 radica

trapping potential; ORAC, oxygen radical absorbance capacity; AUC, area under curve; RMCD, randomly methylated

h-cyclodextrins.* Corresponding author. Tel.: +351 222078968; fax: +351 222004427.

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A. Gomes et al. / J. Biochem. Biophys. Methods 65 (2005) 45–8046

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

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1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2. Fluorescence probes for detection of superoxide radical . . . . . . . . . . . .

2.1. Hydroethidine (dihydroethidium; HE) . . . . . . . . . . . . . . . . . .

2.2. 1,3-Diphenylisobenzofuran (DPBF) . . . . . . . . . . . . . . . . . . .

2.3. 2-(2-Pyridil)-benzothiazoline . . . . . . . . . . . . . . . . . . . . . . .

3. Fluorescence probes for detection of hydrogen peroxide . . . . . . . . . . . .

3.1. 2,7-Dichlorodihydrofluorescein (DCFH) . . . . . . . . . . . . . . . . .

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

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

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

3.5. Dihydrorhodamine 123 (DHR) . . . . . . . . . . . . . . . . . . . . . .

4. Fluorescence probes for detection of singlet oxygen . . . . . . . . . . . . . .

4.1. 9,10-Dimethylanthracene (DMA). . . . . . . . . . . . . . . . . . . . .

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

(DPAXs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

(DMAX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5. Fluorescence probes for detection of hydroxyl radical . . . . . . . . . . . . .

5.1. 4-(9-Anthroyloxy)-2,2,6,6-tetramethylpiperidine-1-oxyl . . . . . . . . .

5.2. 1,3-Cyclohexanedione (CHD) . . . . . . . . . . . . . . . . . . . . . .

5.3. Sodium terephthalate . . . . . . . . . . . . . . . . . . . . . . . . . . .

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

ester of coumarin-3-carboxylic acid (SECCA) . . . . . . . . . . . . . .

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).

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). . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.2. 4,4-Difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-

3-undecanoic acid (C11-BODIPY581/591) . . . . . . . . . . . . . . . . .

6.3. Lipophilic fluorescein derivatives . . . . . . . . . . . . . . . . . . . .

6.4. Dipyridamole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A. Gomes et al. / J. Biochem. Biophys. Methods 65 (2005) 45–80 47

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

6.6. 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) . . . . . . . . . .

6.7. h-Phycoerythrin/Fluorescein/6-Carboxyfluorescein . . . . . . . . . . . .

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/

emission

wavelengths

(nm)

Reactant induced

fluorescence changes

Main reported

applications

HE O2!� 520/610 Production of a red

fluorescent compound

(probably ethidium)

Useful for monitoring the

oxidative burst in cells

DPBF O2!� 410/455

or 477

Fluorescence decrease 1O2 and O2!� detection in

phospholipid liposomes1O2

2-(2-Pyridil)-

benzothiazoline

O2!� 377/528 Production of the fluorescent

2-(2-pyridil)-benzothiazol

Determination of SOD

activity

DCFH H2O2 498/522 Production of the fluorescent

2,7-dichlorofluorescein

The diacetate form

(DCFH-DA) can be applied

in cell studies. Useful as

a marker of the cellular

oxidative stress

HO!

ROO!

Scopoletin H2O2/HRP 360/460 Fluorescence decrease due

to scopoletin’s oxidation

Widely used as an H2O2

monitoring probe, either in

isolated mitochondria, or in

stimulated neutrophils and

eosinophils

Amplex Red H2O2/HRP 563/587 Production of a highly

fluorescent product:

resorufin

Detection of H2O2 in

activated

phagocytic cells as well as in

other types of cells or in

non-cellular systems

HVA H2O2/HRP 312/420 Production of a fluorescent

dimer

Useful for detecting the

production of H2O2 in

isolated mitochondria of

various tissues

DHR H2O2/HRP 505/529 Production of the fluorescent

rhodamine 123

H2O2, HOCl and ONOO�

detection in cells. Evaluation

of scavenging activity

HOCl

DMA 1O2 375/436 Fluorescence decrease by

generation of a non-

fluorescent endoperoxide

Specific 1O2 detection

DPAX 1O2 495/515 Production of a fluorescent

endoperoxide

Specific 1O2 detection

DMAX 1O2 495/515 Production of a fluorescent

endoperoxide

Specific 1O2 detection.

Potential usefulness for

assays in biological systems

4-(9-Anthroyloxy)-

2,2,6,6-

tetramethylpiperidine-

1-oxyl

HO! 377/427 Fluorescence increase

through the elimination of

the intramolecular quenching

Indirect detection of HO!,

involving its reaction with

DMSO and !CH3 production

CHD HO! 400/452 Production of a fluorescent

compound

Indirect detection of HO!,

based on its reaction with

DMSO

Sodium

terephthalate

HO! 310/430 Production of the fluorescent

sodium 2-hydroxy-

terephthalate

Specific HO! detection

(involves hydroxylation

reactions)


Table 1 (continued)

Probe ROS detected Excitation/

emission

wavelengths

(nm)

Reactant induced

fluorescence changes

Main reported

applications

3-CCA and

SECCA

HO! 350 and

395/450

Generation of fluorescent

hydroxylated coumarins

Screening of HO! scavenging

activity and HO! generation

activity

HPF and APF HO! 500/520 Generation of the fluorescent

fluorescein by O-dearylation

Detection of HO! or HOCl

(by combination of the two

probes) in either cellular or

non-cellular systems

HOCl

(only APF)

cis-PnA ROO! 320/432 Irreversible fluorescence

loss upon oxidation

Evaluation of lipid

peroxidation in different

types of cells

C11-BODIPY581/591 ROO! 510/595 Alteration in fluorescence

properties (turns from red

to green)

Evaluation of lipid

peroxidation and antioxidant

efficacy in model

membranes, lipoproteins,

biological fluid

and living cells

RO!

HO!

Lipophilic fluorescein

derivatives

ROO! 495/515 Fluorescence quenching

by hydroperoxides

Determination of lipid

peroxidation associated to

the cellular membrane

Dipyridamole HO! 415/480 Fluorescence decay upon

oxidation

Assessment of the activity of

hydrosoluble or liposoluble

antioxidants

O2!�

ROO!

DPPP ROO! 351/380 Generation of a fluorescent

product: DPPPO

Measurement of the extent of

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

oxidation

Evaluation of the antioxidant

capacity

Fluorescein/

6-carboxyfluorescein

ROO! 495/515 Fluorescence decay upon

oxidation

Evaluation of the antioxidant

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 (kexcitation=520 nm; kemission=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�NFe(II)/H2O2 (i.e. HO!)NO2!�NH2O2. 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

N

CH2CH3

H

NH2H2N

N

CH2CH3

NH2H2N

+

HE E+

λexcitation = 520 nmλemission = 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 (kexcitation=410 nm;

kemission=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

O

λexcitation = 410 nmλemission = 455 or 477 nm

DPBF

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]:

S2O2�4 þ O2 þ 4HO�Y2SO2�

3 þ 2H2O þ O!�2 ð1Þ

2-(2-Pyridil)-benzothiazoline (non-fluorescent), by reaction with O2!�, originates a highly

fluorescent product, 2-(2-pyridil)-benzothiazol (kexcitation=377 nm; kemission=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 (kexcitation=498 nm; kemission=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

HN

N S

N

N S

O2.-


2-(2-Pyridil)-benzothiazoline 2-(2-Pyridil)-benzothiazol

Fig. 3. Oxidation of 2-(2-pyridil)-benzothiazoline by O2!� to 2-(2-pyridil)-benzothiazol (adapted from Ref. [36]).

OH

Cl Cl

OHO

HCOOH

OH

Cl

O

COOH

Cl

O

OCOCH3

Cl Cl

OCOCH3O

HCOOH

DCFH(Non fluorescent)

DCF(Fluorescent)

Oxidation

λexcitation = 498 nm λemission = 522 nm

DCFH-DA(Non fluorescent)

Esteraseor HO-

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

OH

Cl Cl

OHO

HCOO-

HO

Cl

O

COO-Cl

O

OCOCH3

Cl Cl

OCOCH3O

HCOO-

HO

Cl

O

COO

Cl

OH

DCFH

DCF

Esterase or HO-

DCFH-DA

DCF.-

-.

O2O2.-

hv

DCFH DCF.-

-H+

H2O2/Peroxidase

O2

Auto-oxidation

1,3(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 (kexcitation=360 nm; kemission=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

OHO

MeO

O

λexcitation= 360 nm

λemission = 460 nm

Scopoletin

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

(kexcitation=563 nm; kemission=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 Km 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 Km 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 (kmax=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 (N8.3), since de-N-acetylation could occur via nucleophilic substitution and

general base catalysis, resulting in labile dihydroxyphenoxazines [62].

O

N

OHOH

COCH3

O

N

OHO O

N

OHO

Resorufin(Highly fluorescent)

λexcitation = 563 nm

λemission = 587 nm

Amplex Red(Non fluorescent)

O

Resazurin(Non fluorescent)

3

2H2O2 2H2O + O2

1

2

UnknownFluorescent

HRP

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 (z1 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 (kexcitation=312 nm; kemission=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

OH

CH2

MeO

COOH

OH

CH2

MeO

COOH

OH

CH2

COOH

OMe

H2O2

HRP

Homovanillic acid(Non fluorescent)

Fluorescent dimer


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 (kexcitation=505 nm; kemission=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

O NH2H2N

COOCH3H

OH2N

COOCH3

NH2

Dihydrorhodamine 123(Non fluorescent)

Oxidation


λemission = 529 nm

Rhodamine 123(Fluorescent)

+

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 (kexcitation=375 nm, kemission=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 DPAwith 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

CH3

CH3

OO1O2

9,10-Dimethylanthracene Endoperoxide


λemission = 436 nm

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

O OHO

COOH

XX

O OHO

COOH

O

O

XX

X = H DPAX-1 λex= 493 nm λem= 516 nmX = Cl DPAX-2 λex= 507 nm λem= 524 nmX = F DPAX-3 λex= 493 nm λem = 514 nm

1O2

DPAX-1-EP λex= 494 nm λem= 515 nmDPAX-2-EP λex= 506 nm λem= 527 nmDPAX-3-EP λex= 494 nm λem= 515 nm

Fig. 11. Reaction of 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs) with 1O2 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].

H3C

CH3

O OHO

COOH

H3C

CH3

O OHO

COOH

O

O

1O2

DMAX(Low fluorescence)

DMAX-EP(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 (kexcitation=492 nm; kemission=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

ð2Þ

O

O

N CH3

CH3

H3C

H3C

O

O

O

N CH3

CH3

H3C

H3C

O

CH3

Low fluorescence High fluorescence


C

.

.CH3

C

(I) (II)

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

(kexcitation=377 nm; kemission=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 ðCH3Þ2SOYCH3SO2H þ !CH3

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 (kexcitation=400 nm; kemission=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 ðCH3Þ2SOYCH3SO2H þ ! CH3 ð3Þ

!CH3 þ O2YCH3OO! ð4Þ

2CH3OO!YHCHO þ CH3OH 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 (kexcitation6310 nm; kemission6430 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.

OO

N

O O

H

HCHO

NH3


1,3-Cyclohexanedione

Fig. 14. Reaction of 1,3-cyclohexanedione with formaldehyde in the presence of ammonia (adapted from Ref. [83]).

COO-

COO-

COO-

COO-

OH

COO-

O

H

O

O

HO.

C

2-HydroxyterephthalateTerephthalate

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

COOH

O O O

O

O N

O

O

SECCA

C

3-CCA

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 (kexcitation=500 nm; kemission=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

ð6Þ

OOO

COO-

XH

O-OO

COO-

OX

(Almost non fluorescent)

(Strongly fluorescent)


X = O HPFX = NH APF

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 (kexcitation=495 nm, kemission=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 H2O2Yoxidized FLðloss of fluorescenceÞ

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 (kexcitation=320 nm; kemission=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

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 (kexcitation=510 nm; kemission=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,

BF F

N N

(CH2)10-COOH(CH=CH)2

λexcitation = 510 nm λemission = 595 nm

C11-BODIPY581/591

+

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

(kemission=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 (kemission=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 A and a deep pool at b7.5 A 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

NH

HO O O

CO

COOH

NH

HO O O

COOH

S

NHCH2CH2O

O

O

OCH2

C (CH2)14CH3

(CH2)14CH3

O

n = 10 (C11-fluor)n = 14 (C16-fluor)n = 16 (C18-fluor)

(CH2)nCH3

C P

HCO

C

O

H2CO

(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 (kexcitation=415 nm; kemission=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!.

HO

HO

N OH

OH

N

N

N

N

NN

N


Dipyridamole

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 (kexcitation=351 nm; kemission=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: P O


ROOH

DPPP DPPP=O

+ ROH

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 (kexcitation=498 nm; kemission=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 bTotalperoxyl 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 (TTrolox), 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].

ð7Þ


The calculation of the TRAP value is represented by the following equation (adapted from

Ref. [126]):

TRAP ¼ ðTserum =TTroloxÞ � serum dilution factor � 2 � ½Trolox�

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 pHN7, 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 � AUCBlankÞ= ðAUCTrolox � AUCBlankÞ�� 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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