Development of fluorescent and luminescent probes for reactive oxygen species

Accepted Manuscript Title: Development of fluorescent and luminescent probes for reactive oxygen species Author: Huai-Song Wang PII: DOI: Reference:

S0165-9936(16)30208-4 http://dx.doi.org/doi: 10.1016/j.trac.2016.09.006 TRAC 14825

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Trends in Analytical Chemistry

Please cite this article as: Huai-Song Wang, Development of fluorescent and luminescent probes for reactive oxygen species, Trends in Analytical Chemistry (2016), http://dx.doi.org/doi: 10.1016/j.trac.2016.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Development of fluorescent and luminescent probes for reactive oxygen species

1 2 3

Huai-Song Wanga,b

4 5 6 7 8 9

a

Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing, 210009, China.

b

Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of

Education, Nanjing 210009, China.

Highlights

10 11 12 13 14

   

Reactive oxygen species (ROS) can mediate a wide variety of biological processes. Due to their reactive and transient nature, ROS are generally difficult to determination. Fluorescent and luminescent probes for monitoring ROS in biological systems. ROS probes (including small organic molecules, metal complexes and nanomaterials) were discussed.

15 16 17



The design strategies and ROS sensing mechanisms of these functional probes were described.

18

ABSTRACT

19

Reactive oxygen species (ROS) are chemically reactive molecules that can mediate a wide

20

variety of biological processes. The imbalance of these reactive intermediates in the metabolism

21

will result in the phenomenon known as oxidative stress. Therefore, a great number of approaches

22

have been developed for measuring ROS in biological systems. Due to their reactive and transient

23

nature, the ROS are generally difficult to determination. Fluorescent and luminescent probes for

24

monitoring ROS have shown advantages such as high sensitivity, selectivity, as well as real-time

25

imaging, which can yield visible information about the ROS. The recent progress in preparing

26

ROS probes (including small organic molecules, metal complexes or nanomaterials) for detecting

27

and imaging of ROS production in living cells or whole organisms were summarized in this

28

review. The design strategies and ROS sensing mechanisms of these functional probes were

29

described.

30 31

Keywords: Reactive oxygen species; Fluorescent probes; Luminescent probes; Multi-functional

32

probes; Ratiometric probes; Biological systems

33 34

1. Introduction

35

Reactive oxygen species (ROS), as a class of highly reactive chemicals, play important roles in

36

varieties of physiological and pathological processes.[1, 2] The balance of oxidation-antioxidation *

Corresponding author. E-mail: [email protected] (H.-S. Wang) 1

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modulated by ROS in biosystems is crucial for maintaining normal cell functions. That is,

38

ROS-induced disease can be either resulted from the lack of ROS (e.g., chronic granulomatous

39

disease, certain autoimmune disorders) or excessive production of ROS (e.g., cancer, arthritis,

40

arteriosclerosis).[3, 4] ROS can be neutral molecules [such as hydrogen peroxide (H2O2), singlet

41

oxygen (1O2)], ions [such as superoxide (O2•-), hypochlorite (ClO−) and the nitrogen-containing

42

peroxynitrite (ONOO−)] or radicals [such as hydroxyl radical (•OH)].[5, 6] Due to their reactive

43

and transient nature, the ROS are usually difficult to determination especially in biological

44

systems.

45

Most intracellular ROS are derived from the reduction of molecular oxygen in the process of

46

metabolism. The major ROS (O2•- and H2O2) are from the NADPH oxidases (NOXs), xanthine

47

oxidase (XO), and the mitochondrial electron-transport chain.[7-9] Other ROS can be derived

48

from a cascade of transitions from one species to another (Scheme 1), including the superoxide

49

dismutase (SOD) catalyzed formation of H2O2 from O2•-, the reaction of O2•- with •NO to form

50

ONOO-, the peroxidase-catalyzed formation of HOCl from H2O2, and the iron-catalyzed Fenton

51

reaction leading to the generation of •OH.

52

ROS have been considered as key regulatory molecules for cells, but cellular damage can result

53

from the perturbed equilibrium between the formation and transformation of ROS. Due to their

54

high reactivity, ROS readily react with virtually all of the biological molecules. Overproduction of

55

ROS can cause damage to many cellular constituents, including proteins, carbohydrates, lipids,

56

and nucleic acids. Therefore, oxidative stress caused by high-level ROS may be associated with

57

pathologies. The ROS-metabolising systems have become an important research area for better

58

understanding their biological functions.

59 60 61

In the past decades, many approaches (including electrochemical, spectroscopic, and enzymatic

62

techniques) for detecting ROS have been developed.[10-12] The electron spin resonance (ESR)

63

method has been used as a powerful for detecting ROS (such as O2•-, •OH and 1O2), which need to

64

form stable free radicals (spin adducts) by spin trapping.[13, 14] But the ESR technique requires

65

relatively expensive instruments and cannot be employed readily to acquire quantitative estimates

66

of ROS due to many secondary reactions during spin trapping.[15] Therefore, high-performance 2

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liquid chromatography (HPLC) has been employed to separate the byproducts after ROS

68

trapping.[16, 17] The aromatic hydroxylation, arising from the reaction of ROS with aromatic

69

compounds (e.g., salicylic acid,[18] phenylalanine[19] and 4-hydroxybenzoate[20]), is usually

70

used for HPLC method. Several other chemical tools including electrochemical method[21] and

71

mass spectrometry analysis[22] have been devoted in detection of ROS. These methods are still

72

very complex during the sample preparation, which hampers their values for routine analysis.

73

The fluorescent and luminescent probes for monitoring ROS have shown advantages such as

74

high sensitivity, selectivity, as well as real-time imaging, which can yield visible information

75

about the ROS in biological systems.[23-25] In recent years, numerous probes (organic molecules

76

or nanomaterials) have been prepared for selective monitoring one kind of the ROS (such as H2O2,

77

O2•- or •OH) or evaluating the oxidative stress generated from the total ROS. Especially, most of

78

these probes have shown great advantages for in vivo real-time sensing ROS. In this review, I

79

mainly summarize the fluorescent and luminescent probes designed for detecting and imaging of

80

ROS production in living cells or whole organisms.

81 82

2. Different ROS production and the corresponding probes

83

2.1 Superoxide (O2•-) probes

84

The O2•- with highly oxidative activity were considered to be the precursor of other ROS.[26] In

85

the early studies, luminescence- and fluorescence-based assays have been employed to measure

86

cell-derived O2•-. The initial oxidation of the phenanthridine moiety by O2•- to generate a radical

87

intermediate was mostly used for designing the organic probes for O2•-.[27] For example,

88

hydroethidine (HE) and its derivatives were frequently used as probes for sensing local O2•- in the

89

mitochondria. The reaction of the non-fluorescent HE with O2•- leads to a specific hydroxylated

90

product with highly fluorescence (Scheme 2).[28, 29] The major drawback of HE related probes is

91

the poor selectivity toward O2•-. Such probes are usually light-sensitive, and the experiment

92

procedures should be performed in dim light.[5, 30, 31]

93 94 95

Recently, two-photon (TP) fluorescent probes for selectively and sensitively monitoring O2•-

96

were synthesized by Tang and co-workers.[32, 33] Based on the scavenging activity of caffeic acid 3

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(or its derivatives) toward O2•-,[34] the probe 2,5-di(4ʹ-caffeic acid amidestyrene) pyrazine

98

(PY-CA, Scheme 3), conjugated with two caffeic acid molecules and a symmetric styryl-pyrazine

99

as TP absorption cross section, shows high specificity for O2•-, instantaneous response and

100

reversible interaction.[33] The O2•- sensing mechanism is based on the transform from

101

pyrocatechol to benzoquinone. After interacted with O2•-, the generated PY-CAO can emit a

102

maximal fluorescence around 520 nm, and excitation wavelength is 800 nm for TP. This probe can

103

be applied to image O2•- in living tissues and intact organisms. The O2•- images can reach 900 μm

104

into

105

acyl)-1,3,5-triazine-2,4,6- triamine (TCA, Scheme 3), also conjugated with two caffeic acid

106

molecules and show high selectivity for O2•- in live cells and in vivo.[32] Both of the two probes

107

(PY-CA and TCA) exhibit reversible on-off-on type fluorescence response mediated O2•- and

108

glutathione (GSH).

tumor

tissues.

Another

TP

probe,

N,

Nʹ-di-((2E)-3-(3,4-dihydroxyphenyl)acrylic

109 110 111

The cell-penetrating nanotechnology-based probes also offer reliable and durable approach for

112

intracellular biosensing of O2•-. Tian and co-workers prepared a carbon-dot-based probe which

113

employs carbon dots (C-Dots) as the reference fluorophore and HE as the recognition element

114

toward O2•-.[35] The prepared probe CD-HE was used as a dual-emission ratiometric probe: one

115

emission peak at 525 nm from C-Dots as inner reference and one shoulder at 610 nm generated

116

form the reaction between HE and O2•- (Fig. 1). Such inorganic-organic probe demonstrated well

117

stability against pH changes and continuous light illumination and low cytotoxicity. Furthermore,

118

in another work, the HE and fluorescein isothiocyanate (FITC) were loaded in the hollow of the

119

rattle-type silica colloidal particles. The probe with core-shell structure shows similar ratiometric

120

sensing ability toward O2•- compared with the probe CD-HE.[36]

121 122

Even considerable attention has been paid on the development of O2•- sensors, it is still a

123

challenging work to design and synthesize specific and sensitive to O2•-. It might because the

124

instantaneous lifetime and the high oxidation property of O2•-. In biological systems, once O2•- is

125

produced, it will spontaneously or enzymatically changed into H2O2.[37] Therefore, designing

126

fluorescent or luminescent probes that can transiently respond O2•- with well selectivity is still an 4

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important research area.

128 129

2.2 Hydrogen peroxide (H2O2) probes

130

The H2O2 is a relatively stable species comparing with other ROS. Evidence has shown that

131

H2O2 plays an important role as a second messenger in cellular signal transduction.[26, 38] But

132

the generation, degradation, and diffusion of H2O2 are still not well understood.[39] Thus,

133

attentions have been focus on the direct measurement of H2O2 in living systems.

134

2.2.1 Molecular probes for H2O2

135

Boronate-based fluorescent probes, based intramolecular charge-transfer (ICT) mechanism,

136

have been well used to monitor H2O2 in vitro and in vivo, because the reaction of H2O2 with

137

phenyl-boronate is highly selective and faster compared with other ROS.[40, 41] In recent studies,

138

the chemoselective deprotection of boronate esters to phenols was widely used for H2O2 (Scheme

139

4A). Additionally, if the phenyl-boronate is attached to the fluorophore through an ether-linkage

140

(Scheme 4B), a p-quinone-methide will be released after the reaction between phenyl-boronate

141

and H2O2.[42] When the boronate group is removed from the probe, the generated fluorophore

142

with brightly fluorescent can be activated.

143 144

In recent years, the H2O2-mediated “boronate to phenol” conversion is still very popular for

145

designing probes for H2O2 (Fig. 2).[43-46] Most of the probes have good performance in

146

intracellular H2O2 sensing.[40, 47-51] For example, the probe HP-1 consists of a coumarin unit

147

and a diboron xanthene spiro isobenzofuran group bridged by a disulfide bond.[47] It can be used

148

as “turn-on” dual responsive fluorescent probe for the exogenous H2O2 as well as endogenous

149

thiols in living HeLa cancer cells; The HP-3 is an aggregation-induced emission (AIE)

150

fluorescence probe for monitoring H2O2:[49] In the presence of H2O2, the phenylboronic ester

151

moiety can be convert into the phenol group, meanwhile the aggregation will occur based on the

152

AIE because the hydrophilic and hydrophobic properties of the HP-3 is changed.

153 154

Several ratiometric fluorescent probes containing the boronate moiety were also designed for

155

sensing H2O2.[52-56] The ratiometric fluorescent probes can eliminate most ambiguities by two 5

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emission bands that enable an accurate internal calibration. Ihmels and co-workers prepared a

157

series of boronobenzo[b]quinolizinium derivatives allowing ratiometric analysis.[53] Among them,

158

the HP-11 exhibited pronounced light-up effect and higher selectivity towards H2O2 in living cells.

159

According to Fig. 3A, the H2O2 sensing progress can be observed by a continuous decrease of the

160

intensity of the emission maximum around 420 nm which is accompanied by the simultaneous

161

increase of a new redshifted emission band around 540 nm. Hong and co-workers also synthesized

162

a series of styryl dyes in which the formylphenyl boronate esters were used as combinatorial

163

blocks.[54] Especially, the probe HP-12 showed a redshift of about 100 nm at the maximum

164

wavelength upon addition of H2O2 (Fig. 3B). It was successfully utilized for the real-time

165

monitoring of glucose oxidation in the presence of glucose oxidase (GOx).

166 167

Yi and co-workers developed a versatile ratiometric H2O2 probe (HP-13, Fig. 4A) based on

168

1,8-naphthalimide and boric acid ester.[55] The HP-13 exhibited high sensitivity toward H2O2

169

with a fluorescence ratio change of up to 1020-fold. And the HP-13 contains an azide group that

170

makes HP-13 versatile and can be potentially linked to biological molecules via the click reaction.

171

Therefore, it was used to imaging endogenous H2O2. HeLa cells incubated with HP-13 showed

172

strong blue fluorescence at first, then the intensity of the blue fluorescence decreased after

173

interacting with H2O2. Meanwhile, the yellow fluorescence intensity was increased (Fig. 4A).

174

Interestingly, when the HP-13 was modified with Nuclear Localization Signal (NLS) peptide (the

175

transmembrane molecular cargo carrier), the generated probe HP-14 can be delivered into nuclei

176

and ratiometric detection of nuclear H2O2 in living cells (Fig. 4B).

177 178

If the boronate-based fluorescent probe contains an ether- or ester-linkage between the boronate

179

moiety and fluorophore moiety, the reaction between the probe and H2O2 usually trigger the

180

remove of boronate followed by the release of p-quinone-methide.[42, 57, 58] Such H2O2 probes

181

are shown in Scheme 5 and Scheme 6.[59-61] The HP-15 and HP-16 were used as ratiometric

182

fluorescent sensor for H2O2. HP-15 was synthesized based on water-soluble hemi-cyanine dye,

183

which can be easily modified and shows superior photostability.[60] It displayed a colour change

184

from pale orange to pink in the presence of H2O2 with fast response. HP-16 can quantitatively

185

detect H2O2 by ratiometric fluorescence method with a 100 nm red-shifted emission.[61] Thus, 6

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HP-16 can serve as a “naked-eye” probe for H2O2.

187 188

Yin and co-workers designed a chemical probe HP-18 with good stability for detecting

189

H2O2.[62] After interacting with H2O2, the HP-18 exhibited color change from colorless to pink,

190

making it a “naked-eye” probe. The possible mechanism of the reaction of HP-18 with H2O2 was

191

proposed in Scheme 6. This probe is stable under cell culture conditions and can also be used to

192

detect enzymatically generated H2O2. The HP-19 is a turn-on near-infrared (NIR) fluorescent

193

probe for H2O2 based on dicyanomethylene-4H-chromene (DCM) fluorophore.[63] Upon the

194

addition of H2O2, a significant increase in fluorescence intensity at 700 nm can be observed, due

195

to the generation of DCM-OH. The HP-20, as a fluorescence ratiometric sensor molecule, is

196

suitable for trace vapor detection of H2O2.[64] In the presence of H2O2, the fluorescence of HP-20

197

with an emission maximum at 500 nm is converted to an electronic “push-pull” structure (DAT-N),

198

which has an emission peak at 574 nm. The HP-20 exhibits effective vapor sampling of H2O2 with

199

high detection sensitivity (down to 7.7 ppb) and fast sensor response (down to 0.5 s under 1 ppm

200

of H2O2).

201 202

Furthermore, the N-alkylated BODIPY, where p-pinacolborylbenzyl unit was attached through

203

C-N linkage to the fluorophore moiety, has been applied by Shao and co-workers for monitoring

204

and imaging of H2O2 in both living cells and living angelfish.[65, 66] For example, the reaction of

205

HP-21 with H2O2 under physiological conditions can cause the oxidation of the boronate,

206

followed by the 1,6-rearrangement elimination reaction and “Turn-On” fluorescence response with

207

suitable sensitivity (Fig. 5A and B).[66] The probe was used as a mitochondrial-targeting probe

208

for imaging H2O2 in living cells. As shown in Fig. 5C, the HP-21 signal overlaid very well with

209

the fluorescence of Mito Tracker Deep Red (MT DeepRed, commercially available mitochondrial

210

dye). It indicates that the HP-21 was located in the mitochondria and can detect localized H2O2.

211 212

2.2.2 Nanoprobes for H2O2

213

Fluorescent or luminescent nanoparticles have been widely used as fluorescent probes owing to

214

their high photostability and facile surface functionalization for specific targeting. A number of 7

Page 7 of 43

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nanoprobes were recently developed for sensing H2O2 in living cell systems.[25, 67-70] The

216

chemoselectivity of H2O2-induced deboronation also has been applied to the preparation of

217

nanoprobes.[71-74] Niu and co-workers prepared a fluorescence resonance energy transfer

218

(FRET)-based ratiometric nanoprobe to detect intracellular H2O2.[72] The nanoprobe PMT-F127

219

micelle is a self-assembled polymeric micelle, in which the tetraphenylethene (TPE) was selected

220

as the energy donor for FRET and the fluorescent boronate was selected as H2O2-responsive

221

acceptor (Fig. 6A). In the presence of H2O2, the boronate groups were transformed into phenols,

222

resulting in a green-fluorescent fluorescein product (Fig. 6B). In living HeLa cells, the PMT-F127

223

micelles show a blue color at first. When treated with H2O2, the blue color decreased, but green

224

colors increased (Fig. 6C). Such probe was successfully used for FRET-based ratiometric

225

detection of mitochondrial H2O2. In the self-assembly process, several multi-functional molecules

226

such as targeting and energy acceptor moieties can also be simultaneously integrated into the

227

multi-functional nanoprobe.

228 229

Wu and co-workers reported another multifunctional nanoprobe, similar as PMT-F127 micelle,

230

for detecting and imaging mitochondrial H2O2.[74] The nanoprobe possesses both

231

mitochondria-targeting and FRET-based ratiometric sensing capability. The carbon-dot serves as

232

the donor of energy transfer and a boronate-based H2O2 recognition element (PFl) was covalently

233

linked onto CD. In the presence of H2O2, the PFl moieties undergo structural and spectral

234

conversion, affording the nanoplatform a FRET-based ratiometric signal. In the living cell, the

235

nanoprobe can specifically target and stain the mitochondria, as well as track the exogenous H2O2

236

levels.

237

The carbon nanodots (C-dots), as promising fluorescent materials, have elicited much research

238

interest recently due to their outstanding properties, such as good water solubility, biocompatibility,

239

and tunable fluorescence. These merits make the C-dots especially useful for fluorescent

240

bioimaging or biosensing.[75-78] Zhang and co-workers recently designed a C-dots -based

241

fluorescence turn-on sensor employing photo-induced electron transfer (PET) mechanism for

242

H2O2

243

2-(diphenylphosphino)ethylamine, the blue fluorescence of the C-dots was quenched through

244

PET(Fig. 7A and B). In the presence of H2O2, the diphenylphosphine can selectively react with

monitoring

in

aqueous

solutions.[78]

After

being

modified

with

8

Page 8 of 43

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H2O2 and the PET derived from the electron pair of the phosphorus atom to the C-dots was

246

cancelled. Then, the fluorescence intensity of C-dots was increased (Fig. 7C). Chang and

247

co-workers developed a one-pot synthetic strategy for decorating the C-dots on graphene oxide

248

(GO).[77] The prepared C-dots@RGO nanomaterial exhibited well photoluminescence (PL) at a

249

wavelength of 440 nm upon excitation at 365 nm. The H2O2 can quench the photoluminescence of

250

the C-dots@RGO through an etching process. Therefore, such probe was used for detection of

251

H2O2 generated in an acetylcholinesterase (AChE)/choline oxidase (ChOx) system.

252 253

Cerium oxide (CeO2) nanowire was recently found to be a superquencher with long-range

254

energy transfer properties.[79-82] Tang and co-workers prepared a H2O2 nanosensor via binding

255

FAM-tagged single-strand (ss) DNA on CeO2 nanowire (Fig. 8A and B).[79] The Ce4+ on the

256

surface of CeO2 exhibits well DNA binding affinity by means of metal coordination. After

257

assembling the CeO2 nanowires with FAM-labeled ssDNA, the fluorescence of FAM groups was

258

quenched. Upon addition of H2O2, the FAM-labeled ssDNA was released form CeO2 nanowires

259

followed by fluorescence signal increasing. The designed CeO2-DNA nanosensor is capable of

260

rapidly (<1 min) and selectively tracking H2O2 in living cells and zebrafish larvae (Fig. 8C and D).

261

Qu and co-workers synthesized a H2O2 nanosenser by doping the Eu3+ into CeO2 nanorods.[81]

262

The fluorescence emission peaks can be assigned to the 5D0-7F J (J = 0-4) transitions of Eu3+ ions.

263

After treating with L(+)-ascorbic acid, the fluorescence intensity of the Eu3+-doped CeO2 nanorods

264

was decreased, resulting from the chemical reduction of Ce4+ to Ce3+ by ascorbic acid and

265

subsequent excitation cut-off of charge transfer from O2- to Ce4+. Interestingly, the fluorescence

266

could be completely recovered to their original intensity when the reduced samples were treated

267

by H2O2. Such Eu3+-doped CeO2 nanorods can be used as fluorescence switcher by alternatively

268

adding ascorbic acid and H2O2.

269 270

Graphene quantum dot (GQD) has shown great promise in the field of biosensing due to its

271

photoluminescence property contributed by quantum confinement and edge effects. Yang and

272

co-workers prepared a kind of AgNP/GQDs hybrid nanocomposite for high performance H2O2

273

detection (Fig. 9A).[83] In such nanocomposite, Ag NPs acted as quencher and recognition unit,

274

and GQDs served as a signal output unit with excellent optical property. The GQDs were 9

Page 9 of 43

275

assembled on the surface of ssDNA-modified AgNPs through π-π stacking (Fig. 9B). Then, the

276

fluorescence of GQDs was quenched by Ag NPs through the resonance energy transfer. Upon

277

H2O2 addition, obvious fluorescence recovery can be observed due to the Ag NPs etching and

278

DNA cleavage. The reaction between H2O2 and Ag NPs can generate •OH that will cleave the

279

DNA-bridge and result in the disassembly of AgNP/GQDs with further signal enhancement (Fig.

280

9C).

281 282

The functionalized fluorescent metal nanoclusters with well quantum yield have been used to

283

determine H2O2.[84, 85] For example, Lu and co-workers used the gold nanoclusters as the model

284

aggregation-induced

285

bis(2,4,6-trichlorophenyl) oxalate (TCPO)-H2O2 chemiluminescence (CL) reaction.[85] This

286

research shows that the unique AIE effect of gold nanoclusters can strongly boost the CL signal of

287

the TCPO-H2O2 system, due to the chemiluminescence resonance energy transfer (CRET) between

288

TCPO energy donors and gold nanocluster aggregate acceptors. Additionally, another CRET based

289

H2O2 probe, constructed by the assembly of CdTe quantum dots (QDs) upon the surface of layered

290

double hydroxide (LDH), has been prepared by Lu and co-workers.[86] The H2O2 recognition is

291

based on the luminol-H2O2 system, and the oriented QD-LDH nanocomposite can efficiently

292

accept the energy from luminol donors for signal amplification. This method exhibited a stable

293

response to H2O2 with a detection limit as low as 0.3 μ M.

emission

(AIE)

molecules

to

study

their

influence

on

the

294 295

2.3 Hydroxyl radical (•OH) probes

296

The •OH is generally considered as the most aggressive radicals among the ROS.[87, 88] It can be

297

generated within cells by the Fenton reaction enabled by transition metals, and plays a critical role

298

in numerous pathological processes. Different methods have been developed for the indirect or

299

direct detection of •OH, including electron paramagnetic resonance (EPR), chemiluminescence,

300

HPLC method, UV-vis spectroscopy and fluorescence methods.[89] In the past decades, the

301

fluorescent probes for •OH have been widely reported employing organic dye molecules or

302

fluorescent nanoparticles (quantum dots, metal nanoclusters, etc.). But, designing biocompatible

303

fluorescent probes for accurate and selective detection and quantification of •OH is still a

304

challenge. 10

Page 10 of 43

305

The spin labeled fluorescent probes (R2-NO•), such as hydroxyl-2,2,6,6-tetramethylpiperidine

306

-N-oxide (TEMPO), have been used for trapping and detecting the •OH. The fluorescence of the

307

fluorophore can be quenched by the nitroxide group through electron exchanges, and can be

308

recovered after interacted with a free radical.[90] For example, the probe HR-1 (Scheme 7) was

309

recently applied for trapping the •OH, then quantitatively detected by HPLC).[91] This probe has

310

the potential to trace •OH production induced by the impairment of the mitochondrial respiratory

311

chain.

312 313

Tae and co-workers prepared a fluorescent probe (HR-2, Scheme 8) for monitoring •OH based

314

on the oxidative C-H abstraction reaction of rhodamine cyclic hydrazide.[92] The •OH-induced

315

reaction of HR-2 takes place rapidly at room temperature and opens the spirocyclic ring system.

316

The probe exhibited excellent selectivity for monitoring intracellular •OH with virtually no

317

interference by other ROS species. Pierre and co-workers prepared a •OH probe (HR-3) that

318

consisted of a terbium complex with open coordination sites and a reactive pre-antenna composed

319

of an aromatic acid.[93] Without •OH, the trimesate does not coordinate, and the emission of the

320

terbium ion was not sensitized. After the reaction with •OH, the hydroxylated trimesate was

321

readily bind to terbium complex, and the terbium-centered emission was increased. Yi and

322

co-workers developed a naphthalimide-naphthyridine derivative (HR-4) for the detection of

323

•OH.[94] The reaction between HR-4 and •OH can generate a hydroxyl modified naphthyridine

324

moiety with blue fluorescence emission. The HR-4 shows excellent photostability, low

325

cytotoxicity and high biocompatibility. It shows no cellular toxicity in 36 h at a concentration of 5

326

mM.

327 328

Au (or Ag) nanoclusters have attracted significant attentions as luminescent nanomaterials due

329

to the strong quantum-confinement effect.[95, 96] Several works have reported for detecting •OH

330

based on Au or Ag nanoclusters.[97, 98] Tian and co-workers prepared a ratiometric fluorescence

331

biosensor, employing Au nanocluster (AuNC) protected by bovine serum albumin (BSA) as

332

reference fluorophore, which was conjugated with HPF (HR-5) as both the response signal and

333

specific recognition element for •OH (Fig. 10A).[98] The prepared AuNC@HPF probe showed

334

only one emission peak at 637 nm ascribed to AuNCs without •OH, because the HR-5 was almost 11

Page 11 of 43

335

non fluorescent. After interacted with •OH, the fluorescence emission at 515 nm was gradually

336

increased due to the oxidation of HR-5 (Fig. 10B), while the emission at 637 nm stays constant.

337

For ratiometric determination of •OH generated in cells, lipopolysaccharides (LPS) was

338

introduced to induce oxidative stress. The results show that the ratiometric sensor was successfully

339

used for bioimaging and monitoring of the •OH changes.

340 341

The C-dots have also been sued for sensing •OH.[99] By employing the polyvinylpyrrolidone

342

(PVP) as the only carbon source, the prepared C dots exhibit good photostability in a wide range

343

of pH solution (from 3.0 to 10.5) and excellent water solubility. Interestingly, the fluorescence

344

intensity of the C dots can be sensitively affected by •OH.

345 346

2.4 Singlet oxygen (1O2) probes

347

Recent researches have shown that the chemistry of 1O2 exhibited large biomedical significance. In

348

the medical studies, the 1O2 plays a key role in photodynamic therapy (PDT), an emerging

349

anticancer treatment using photoirradiation and photosensitizers (Sens).[100, 101] During the PDT,

350

the Sens can transfer the absorbed energy to molecular oxygen to generate 1O2, which can finally

351

destroy the cancer cells.[102] However, the 1O2 can also lead to some diseases because it is prone

352

to destruct the biological molecules (such as proteins, nucleic acids and lipids).

353

There have been several fluorescent probes for monitoring for 1O2 further outstanding

354

importance of 1O2 in photobiological processes.[103] Chemical probes, based on the reaction

355

between 1O2 and the derivatives of anthracene to form stable endoperoxides, were most frequently

356

used.[104-107] The probes with anthracene moiety can react with 1O2 via the highly favorable [4

357

+ 2] cycloaddition mechanism. A typical anthracene based 1O2 probe was shown in Scheme 9: in

358

the presence of

359

anthraquinone were broken, followed by rapid fluorescence recovery of the fluorophores.[108]

1

O2, the linkers between fluorophores (F1 and F2) and the generated

360 361 362

Majima and co-workers recently developed a far-red fluorescence probe (SO-1, Fig. 11A) for

363

monitoring 1O2 based on 9,10-diphenylanthracene (DPA).[109] The SO-1 is composed of

364

silicon-containing rhodamine and an anthracene moiety as 1O2 reactive site. It exhibited a good 12

Page 12 of 43

365

selectivity toward 1O2 out of other ROS with the emission wavelength at 640 nm (Fig. 11B). In the

366

cell experiment, 5-aminolevulinic acid (ALA) was taken up by cancer cells and then metabolized

367

to the heme precursor protoporphyrin IX (PpIX) to become a photosensitizer. The photoirradiation

368

of PpIX can generate 1O2, meanwhile increase the fluorescence intensity of SO-1 rapidly (within

369

10

370

[tetra-(N-methyl-4-pyridyl)porphyrin, TMPyP4], which changes its location from lysosome to

371

cytoplasm and nucleus upon photoirradiation (Fig. 11C). The results proved that the SO-1 can

372

respond to only mitochondrial-originated 1O2.

s).

The

SO-1

does

not

react

with

1

O2

generated

by

another

Sens

373 374

Some luminescent probes were also designed for monitoring 1O2 during the photodynamic

375

therapy.[110, 111] The SO-2 in Scheme 10 is a Eu3+ complex-based luminescence, in which the

376

terpyridine moiety is an antenna for sensitizing the Eu3+ luminescence, and the

377

10-methyl-9-anthryl moiety plays the roles of quenching the luminescence of Eu3+ and selectively

378

trapping 1O2.[110] The SO-2 and PDT drugs, indole-3-acetic acid (IAA) and hematoporphyrin

379

monomethyl ether (HMME), were co-loaded in HeLa cells. The SO-2 can react with the generated

380

1

381

lifetime. The SO-3, constructed based on a fluorescent coumarin group and a phosphorescent Ir3+

382

complex, is a ratiometric luminescent probe for monitoring 1O2.[111] The mechanism for

383

monitoring 1O2 is based on the convering the julolidine of coumarin group to an iminium form

384

(Scheme 10). The SO-3 was successfully used to monitor therapeutic 1O2 dosages by recording

385

the ratiometric photoluminescence changes.

O2 to form its endoperoxide followed by the remarkable increases in luminescence intensity and

386 387 388

2.5 Hypochlorite (ClO−) probes

389

The hypochlorite/hypochlorous acid (HOCl/ClO−) exerts a wide variety of physiological effects in

390

living systems. Endogenous HOCl/ClO− can be produced from the reaction of H2O2 and chloride

391

ions catalyzed by myeloperoxidase (MPO). The function of HOCl/ClO− in physiological processes

392

is mainly for protecting the body against microorganism invasion.[112-114] However, the

393

uncontrolled HOCl/ClO− production may lead to many inflammation-related diseases.[115]

394

A number of probes for visualizing HOCl/ClO− in intercellular systems have been repored.[116] 13

Page 13 of 43

395

Recently, a visible-light-excitable fluorescence ratiometric probe for ClO− was synthesized by

396

Goswami and co-workers.[117] The probe (H-1) exhibits an emission peak at 630 nm when

397

excited at 430 nm. The H-1 can be oxidatively attacked by ClO− to the imino group, which later

398

may lose the diaminomeleonitrile unit (Scheme 11). The final product exhibits a new emission

399

peak at 485 nm with the peak decrease at 630 nm.

400 401 402 403

Wang and co-workers developed a near-infrared fluorescent probe (H-2, Scheme 12) for HOCl

404

synthesized by activating the polymethine chain of a cyanine dye.[118] The reaction between H-2

405

and HOCl can result in the NIR fluorescence quenching at 774 nm. The reaction mechanism was

406

confirmed by mass spectra as electrophilic addition to the polymethine chain, then followed by

407

oxidation cleavage. Yuan and co-workers prepared a ruthenium(II) complex-based phosphorescent

408

probe (H-3) for HOCl.[119] In the probe, the Ru(II) complex was used as the signaling unit and an

409

amide linkage as the specific reaction moiety for HOCl. The oxidation reaction of the amide

410

linkage can be promoted by HOCl, and the generated -N-Cl species can further undergo a

411

hydrolysis process to form a highly luminescent complex.

412 413

The probe H-4 is a ratiometric fluorescence sensor for HOCl based on FRET form coumarin

414

moiety to rhodamine moiety.[120] In the absence of HOCl/ClO−, only donor (coumarin moiety)

415

fluorescence emission can be observed at 470 nm. After interacted with HOCl/ClO−, the

416

thiohydrazide spiro-ring opening reaction was occurred, meanwhile the fluorescence intensity at

417

470 nm diminished and a new emission of the acceptor at 580 nm appeared. The probe H-5

418

contains a coumarin fluorophor and an arylseleno moiety.[121] The HOCl/ClO− sensing

419

mechanism is based on the selenoxide elimination followed by a significant fluorescent turn-on

420

signal. It can rapidly respond to HOCl/ClO− within seconds with well selectivity.

421 422

2.6 Peroxynitrite (ONOO−) probes

423

Peroxynitrite (ONOO−) is formed from the reaction of nitric oxide (•NO) with superoxide O2•- in

424

inflammatory cells such as neutrophils and macrophages. The well-controlled generation of 14

Page 14 of 43

425

ONOO− is involved in cell signal transduction. As a strong oxidant, the ONOO− can also react

426

directly with a wide array of biomolecules (e.g., proteins and DNA). Therefore, sensitive and

427

selective methods (such as using fluorescent and luminescent probes) have been developed for

428

better understanding of the role played by ONOO− in cellular functions.

429

Recent investigations have shown that some boronate-containing fluorogenic compounds can

430

selectively react with ONOO− to yield corresponding hydroxyl derivatives.[122-124] Based on

431

such reaction, several fluorescent probes were designed for detecting ONOO−. Han and

432

co-workers synthesized an ONOO−probe (P-1) composed of pyrene dye and a dioxaborolane

433

group (Scheme 13).[123] In the faintly fluorescent P-1, the boronate, with sp2-hybridized boron

434

atom, is very intensely Lewis acidic. The ONOO− as Lewis base can attack the boron atom eagerly

435

and form a peroxyborate intermediate. Then aryl migration and quantitative hydrolysis give the

436

fluorescent phenol product. Another boronate-based fluorescent probe (P-2) was synthesized by

437

Kim and co-workers.[124] The boronate probe P-2 contains a phenol moiety masked by a

438

p-dihydroxyborylbenzyloxy reaction site, and displays a low fluorescence quantum yield. After

439

the reaction with ONOO−, the arylboronate group will be oxidized to its corresponding phenol,

440

which would undergo rapid elimination of p-quinomethane to produce fluorescent phenolate.

441 442

The P-3 is a ruthenium(II) complex-based fluorescent sensor for ONOO−.[125] The

443

aryloxyphenol group of P-3 was used as the ONOO− accepting unit. The reaction mechanism

444

between P-3 and ONOO− was confirmed by MS and NMR spectra. The addition of to the P-3

445

solution can result in distinct fluorescence quenching at 600 nm due to the O-dealkylation reaction.

446

The probe shows favorable water-solubility, biocompatibility and rapid reaction with ONOO−.

447

James and co-workers developed a probe via self-assembling aromatic boronic acids with

448

alizarin red S (ARS) for colorimetric and fluorometric detection of ONOO−.[126] A boronic acid,

449

2-(N,N-dimethylaminomethyl)phenylboronic acid (NBA), was successfully assembled with ARS

450

(Scheme 14). The assembling of ARS with NBA results in two species (one major and one minor

451

with approximately 2 : 1 ratio), which were used as ONOO− probe (P-4). The ONOO− can react

452

with boronate-based P-4 to produce the phenol analogues and lead to the release of ARS,

453

meanwhile giving a fluorescence decrease and color change.

454 15

Page 15 of 43

455

Yoon and co-workers designed and synthesized another colorimetric and fluorometric probe P-5

456

(Scheme 15) for ONOO−.[127] The P-5 contains a hybrid coumarin–hemicyanine scaffold. The

457

interaction between P-5 and ONOO− was monitored by using NMR and mass spectrometry, which

458

shows

459

coumarin-3-aldehyde. In this process, the emission peak of P-5 at 635 nm decreases in concert

460

with an increase of a new and more emissive band at 515 nm, and the color of the solution was

461

changed from blue violet to faint yellow.

the

final

products

consisting

of

predominantly

1,3,3-trimethyloxiindole

and

462 463

Yang and co-workers designed and synthesized a rhodamine-based ONOO− probe (P-6, Fig.

464

12A).[128] The probe P-6 was non-fluorescent. In the presence of ONOO−, the ONOO− triggered

465

N-dearylation reaction can be occurred followed by fluorescence turn-on response. The P-6

466

derivative (P-7, Fig. 12B) with a caged carboxylate is neutral and can readily diffuse across the

467

cell membrane. Therefore, the P-7 was applied to image the generation of endogenous ONOO− in

468

living cells and issues. As shown in Fig. 12C, the P-7 exhibited high sensitivity and selectivity

469

toward ONOO−.

470 471 472

Sevral chemiluminescence probes for ONOO− were recently developed by Lu and

473

co-workers.[129, 130] Typically, the thioglycolic acid (TGA)-capped CdTe QDs was used for

474

specific detection of ONOO− in living cells.[130] Generally, the ONOO− can decompose into

475

oxidizing and reducing radical pair, which can interact with QDs to produce the CL emissions by

476

electron-transfer annihilation. It was found that the oxidizing radical •OH from ONOOH can

477

inject a hole into the valence band (VB) of the CdTe QDs to produce the oxidized QDs (QDs +).

478

Then, electron-transfer annihilation between QDs + and O2•- (form ONOO−) was occurred followed

479

by light emission. This probe features an excellent selectivity for ONOO−, and it is the first

480

chemiluminescence probe available for the detection of ONOO− in living cells.

•

•

481 482

2.7 Multi-ROS probes

483

Multiple ROS including (H2O2, 1O2, O2•-, ClO−, ONOO− and •OH) generally coexist in

484

physiological progresses. Because the total ROS level has been considered to be one of the major 16

Page 16 of 43

485

characteristics of many diseases, several methods for detecting ROS level have been recently

486

developed using fluorophores, luminophores and quantum dots.[131-134] Over the past decades

487

years, one of the most commonly used fluorescent probes for ROS is the non-fluorescent

488

2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA).[135, 136] DCFH-DA can easily pass the

489

cell membrane and is cleaved by intracellular esterases to 2',7'-dichlorodihydrofluorescein

490

(DCFH). The DCFH is then oxidized by ROS to the highly fluorescent dichlorofluorescein (DCF).

491

Recent years, the research attentions mainly focus on designing nano-probes for ROS level. The

492

Au nanoclusters (AuNCs) synthesized using glutathione template have been used as biosensing

493

substrates for ROS sensing.[137, 138] Typically, Chu and co-workers developed a nanocomplex

494

displaying single-excitation and dual-emission fluorescent properties towards highly reactive

495

oxygen species (hROS), including ClO−, ONOO− and •OH.[138] The nanocomplex is an

496

AuNC-decorated silica particle, in which CF 405S succinimidyl ester (an amine-reactive

497

fluorescent dye with a strong and photostable emission peak) is located in silica nanoparticle and

498

the glutathione templated AuNCs are grafted on the silica surface via streptavidin (SA)-biotin (Fig.

499

13A and B). The nanosensor exhibits two fluorescence peaks: one located at 435 nm arising from

500

CF 405S, and the other at 565 nm originating from the decorated AuNCs. It can selectively react

501

with hROS followed by fluorescence quenching at 565 nm, and shows excellent biocompatibility

502

for sensing of hROS in living cells (Fig. 13C and D).

503 504

The AuNCs synthesized using glutathione template can also assembled with C-dots as hROS

505

sensor.[139] When the AuNCs and C-dots are fabricated in nanoparticle (termed as C-dots-AuNC),

506

the fluorescence of AuNCs was enhanced while the fluorescence of C-dots was stabilized.

507

Interestingly, the fluorescence of AuNCs can be quenched in response to hROS, but the

508

fluorescence from the C-dots negatively decreased. Therefore, such dual-emission property of

509

C-dots-AuNC allows sensitive imaging and monitoring of the hROS signal (Fig. 14).

510 511

There have been several works reported by using C-dots for directly sensing ROS.[140, 141]

512

The phosphorus and nitrogen doped carbon dots (PN-CDs) were prepared by carbonization of

513

adenosine-5′-triphosphate using a hydrothermal treatment.[141] The PN-CDs shows rapid

514

fluorescence quenching in the presence of ROS (especially the ClO−), and can be applied for 17

Page 17 of 43

515

label-free, sensitive and real-time detection of ROS.

516

Fluorescent coordination polymers, a class of hybrid materials assembled from polydentate

517

bridging ligands and metal ions, have shown their excellent properties for sensing ROS. Such

518

materials possess several potential advantages such as structural and chemical diversity and their

519

intrinsic biodegradability.[142] Very recently, xia and co-workers designed a sulfur-tagged

520

europium(III) coordination polymers for monitoring ROS in living cell and aerosols.[143] The

521

probe were prepared by simply mixing the bridging ligand (2,2′-thiodiacetic acid) and Eu3+ in

522

ethanol. The product morphology can be transformed from microcrystals (at 25 ºC) to

523

nanoparticles (at 150 ºC) upon increasing reaction temperature. In the presence of ROS, the

524

emission peaks of Eu3+ are quenched due to the thioether groups of the ligand are oxidized to

525

sulfoxide followed by intramolecular charge transfer from sulfoxide to Eu3+ (Fig. 15).

526 527 528

3.

Conclusion

529

The development of fluorescent and luminescent probes for ROS in recent three years was

530

discussed in this review. Up to now, the great challenge of designing ideal ROS probe is still the

531

selectivity and sensitivity when used in real complex systems, especially in biological samples and

532

living cells. In the living systems, the low concentrations and short lifetimes have hampered the

533

detection of most of ROS, including H2O2, 1O2, O2•-, ClO−, ONOO− and •OH. Furthermore, the

534

traditional fluorescent probes generally have some shortcomings (e.g., background interference,

535

easy to be photobleached and so on). Recently, many probes based on small organic molecules or

536

metal complexes have shown good selectivity toward different kinds of ROS, such as the

537

boronate-based fluorescent probes for H2O2 and DPA-based fluorescent probes for

538

Nevertheless, compared with the probes for H2O2, there are few probes specially designed for

539

sensing 1O2, O2•-, ClO− or ONOO− in recent 3 years.

1

O2.

540

The emerged nanomaterials have provided promising sensing platforms for ROS, because of

541

their unique optical and catalytic properties for translating the biorecognition events to

542

spectroscopic responses. Modification of the surface of such nanomaterials with biomolecules,

543

such as antibodies or peptides, can reduce their cytotoxicity, facilitate their internalization into

544

cells. Therefore, the multifunctional fluorescent nano-probes have shown high potential in the 18

Page 18 of 43

545

field of intracellular ROS sensing. Furthermore, Most of the nanocomposites can be designed as

546

dual-colored ratiometric nanoprobes for more accurately monitoring ROS. And designing

547

near-infrared nano-probes will facilitate the in vivo ROS sensing with low background

548

interference and high penetrability into tissues.

549

Generally, the intracellular ROS level was very high in in pathological processes including

550

cancer, neurodegenerative injury and inflammation. In recent years, many researchers are focus on

551

designing multifunctional nanoprobes with property of ROS sensing combined with ROS-induced

552

drug release, thus offering a step toward the development of theranostic nanomedicines. Designing

553

smart nanomaterials provide a compelling approach for the future development of ROS probes for

554

bioimaging and therapeutic applications.

555 556

Acknowledgements

557

This work was supported by Jiangsu Provincial Natural Science Foundation (No. BK20150689

558

and

559

BE2016745), the Open Project Program of MOE Key Laboratory of Drug Quality Control and

560

Pharmacovigilance (No. DQCP2015QN01)，the National Natural Science Foundation of China

561

(Grants 81673390) and the Fundamental Research Funds for the Central Universities (No.

562

2015PY010 and No. 2015ZD008).

No.

BK

20151445),

Jiangsu Provincial Key Research and Development Program (No.

563 564

References

565 566 567 568 569 570 571 572 573 574 575 576 577 578

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910 911

27

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Fig. 1. Carbon-dot-based fluorescent probe for imaging O2•-. (A) Working principle of the O2•- sensing. (B and C)

914

Pseudocolored ratiometric images of HeLa cells containing CD-HE probes before (B) and after (C) induced by

915

LPS (a stimulator for production of ROS). Reproduced with permission of the American Chemical Society

916

from ref.[35]

917

918 919

Fig. 2. Structures of H2O2 probes based on “boronate to phenol” conversion.

920

28

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921 922

Fig. 3. Fluorimetric monitoring H2O2 with HP-11 and HP-12. Arrows indicate the development of emission bands

923

with time (A) or with various concentrations of H2O2 (B). Reproduced with permission of The Royal Society of

924

Chemistry from ref.[53] and Wiley-VCH from ref.[54]

925

926 927

Fig. 4. Ratiometric fluorescent probes for monitoring cytoplasmic and nuclear H2O2. (A) Confocal laser scanning

928

microscopy (CLSM) ratio (RY/B) images of 5 μM HP-13-loaded HeLa cells stimulated with 200 μM H2O2 for

929

(a) 0, (b) 30, (c) 60 min at 37 °C. (A) CLSM ratio (RY/B) images of 50 μM HP-14-loaded HeLa cells stimulated

930

with 200 μM H2O2 for (a) 0, (b) 30, (c) 75 min at 37 °C. RY/B was constructed by fluorescence detection at

931

yellow channel and blue channel. Reproduced with permission of the American Chemical Society from ref.[55]

932

29

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933 934

Fig. 5. Mitochondria-targeted fluorescent probe for H2O2. (A) The probe HP-21 and its reaction with H2O2. (B)

935

The fluorescence spectra of probe HP-21 (5 μM, black line) and the reaction mixture (red line) of 5 μM probe

936

HP-21 with 50 μM H2O2. (C) Confocal fluorescence images of HeLa cells incubated with HP-21: C1,

937

probe-stained HeLa cells treated with 100 μM H2O2 for 90 min; C2, co-staining and imaged with 50 nM MT

938

DeepRed; C3, Merged images of C1 and C2. Reproduced with permission of the American Chemical Society

939

from ref.[66]

940

941 942

Fig. 6. Polymeric nanoprobes for FRET-based ratiometric detection of H2O2. (A) Schematic illustration for 30

Page 30 of 43

943

micelle-based ratiometric sensing of mitochondrial H2O2 in a living cell. (B) Fluorescence emission spectra of

944

PMT-F127 nanoprobe. (C) Confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with

945

PMT-F127 micelles with the addition of 0 µM (control, C1), 50 µM (C2) and 200 µM (C3) H2O2. Reproduced

946

with permission of The Royal Society of Chemistry from ref.[72]

947

948 949

Fig. 7. Carbon dot-based fluorescence turn-on sensor for H2O2. (A) Schematic of the sensing process for H2O2. (B)

950

TEM image of the C-dots. The insets show the particle size distribution histogram (n = 60) and HRTEM image

951

of the C-dots. (C) Fluorescence spectra of the nanoprobes with the gradual addition of H2O2. Reproduced with

952

permission of The Royal Society of Chemistry from ref.[78]

953

954 955

Fig. 8. CeO2 nanowire-DNA nanosensor for H2O2. (A) Competitive coordination mechanism of the CeO2 31

Page 31 of 43

956

nanowire-DNA nanosensor for H2O2 detection. (B) TEM images of CeO2 nanowire. (C) Real-time imaging and

957

quantification of H2O2 by CeO2 nanowire-DNA nanosensor using imaging flow cytometry. (D) Real-time

958

fluorescence imaging of H2O2 production in zebrafish larvae. Reproduced with permission of The Royal

959

Society of Chemistry from ref.[79]

960

961 962

Fig. 9. AgNP/GQDs hybrid nanocomposite for H2O2 detection. (A) Schematic description of H2O2 based on

963

AgNP/GQDs. (B) and (C) TEM image of AgNP/GQDs before (B) and after (C) H2O2 addition. Reproduced

964

with permission of the American Chemical Society from ref.[83]

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Fig. 10. Ratiometric fluorescence probe for monitoring •OH in live cells based on gold nanoclusters. (A) Working

968

principle of the AuNC@HPF probe for •OH detection. (B) Reaction scheme of HPF with •OH. (C1) The

969

overlay of confocal fluorescence image and the bright-field image of Hela cells before being exposed to •OH.

970

(C2 and C3) The ratio images of Hela cells with AuNC@HPF probe after being stimulated by LPS for 45 and

971

90 min, respectively. Reproduced with permission of the American Chemical Society from ref.[98]

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Fig. 11. Far-red fluorescence probe for monitoring 1O2. (A) The reaction between SO-1 and O2. (B) Selectivity of

975

SO-1 toward 1O2 among other ROS. (C) Fluorescence images of HeLa cells incubated with (a) SO-1 and

976

5-ALA-induced PpIX, and with (b) SO-1, TMPyP4 and lysosome marker. Reproduced with permission of the

977

American Chemical Society from ref.[109]

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979 980

Fig. 12. Rhodamine-based fluorescent probe for molecular imaging ONOO−. (A) The reaction between P-6 and

981

ONOO− to yield a strongly fluorescent product. (B) The structure of cell membrane permeable P-7. (C) The

982

ONOO− imaging in SH-SY5Y human neuroblastoma cells. The cells were incubated with HKYellow-AM

983

firstly, and then treated with H2O2 or the indicated ROS donors for 1 h, followed by fluorescence imaging.

984

NOC-18, MSB, and SIN-1 were used to produce •NO, O2•-, and ONOO− respectively, FeTMPyP was used as an

985

ONOO− decomposition catalyst. The scale bar was 20 μm. Reproduced with permission of The Royal Society

986

of Chemistry from ref.[128]

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988 989

Fig. 13. AuNC-decorated silica particles for live cell imaging of hROS. (A) Schematic illustration for hROS by

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AuNC-decorated silica particles. (B) TEM image of AuNC-decorated silica particles. (C) Ratiometric

991

responses of the nanosensor for hROS. (D) Confocal fluorescence microscopy images of HL-60 cells treated

992

with (a) no stimulation and (b) H2O2 for 10 min after incubating with AuNC-decorated silica particles.

993

Reproduced with permission of the American Chemical Society from ref.[138]

994

995 996

Fig. 14. C-dots-AuNC nanocomplex for hROS sensing. (A) Schematic illustration of the construction of

997

C-dots-AuNC and the working principle for detecting hROS. (B) TEM image of C-dots-AuNC (Red circles

998

and blue circles represent AuNCs and C-dots, respectively). (C) Fluorescence spectra of C-dots-AuNC in the 36

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999

presence of ClO-. Reproduced with permission of the American Chemical Society from ref.[139]

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1001 1002 1003

Fig. 15. Fluorescent sulfur-tagged europium(III) coordination polymers for monitoring ROS. Reproduced with permission of the American Chemical Society from ref.[143]

1004 1005

1006 1007

Scheme 1. Typical progresses of generation and transformation of the intracellular ROS.

1008

1009 1010

Scheme 2. Proposed mechanism of the oxidation of HE by O2•-.

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1012 1013

Scheme 3.The structure and luminescence mechanism of the two-photon fluorescence imaging probes.

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1015 1016

Scheme 4. The reaction mechanism of the boronate-based fluorescent probes for H2O2.

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1018 1019

Scheme 5. The reaction of the boronate-based fluorescent probes for H2O2.

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1021 1022

Scheme 6. The reaction of the selected boronate-based probes for H2O2.

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1024 1025

Scheme 7. The detection mechanism for •OH by HR-1.

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1027 1028

Scheme 8. The reaction of fluorescent probes for •OH.

1029

1030 1031

Scheme 9. The reaction mechanism of the anthracene-based fluorescent probes for 1O2.

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40

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1033 1034

Scheme 10. Reactions of luminescent probes with 1O2.

1035

1036 1037

Scheme 11. Possible mechanism of the response of H1 towards HOCl/ClO−.

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1039 1040

Scheme 12. Reactions of sensors for HOCl/ClO− detection.

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1042 1043

Scheme 13. Reactions of sensors for ONOO− detection.

1044

1045 1046

Scheme 14. Sensing mechanism of P-4 complex probe for ONOO−.

1047

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1048 1049

Scheme 15. Propose sensing mechanism of P-5 for ONOO−.

43

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