Advances in functional fluorescent and luminescent probes ... · PDF fileand luminescent...

Trends in Analytical Chemistry, Vol. 39, 2012 Trends

Advances in functional fluorescentand luminescent probes for imagingintracellular small-moleculereactive speciesShiguo Wang, Na Li, Wei Pan, Bo Tang

We summarize recent progress in imaging intracellular small-molecule reactive species (ISMRS) by functional probes. In mol-

ecular imaging, functional fluorescent and luminescent probes (e.g., ratiometric, targetable fluorescent, reversible fluorescent,

and multi-functional) and corresponding nanoprobes have great potential for investigating ISMRS-mediated cell-signal transd-

uction. We describe design strategies for the development of functional probes. Future research on ISMRS will benefit from

recent advances in the development of new functional probes for selective detection of ISMRS.

ª 2012 Elsevier Ltd. All rights reserved.

Keywords: Intracellular small-molecule reactive species (ISMRS); Fluorescent probe; Luminescent probe; Molecular imaging; Multi-functional

probe; Nanoprobe; Ratiometric probe; Reversible probe; Targetable probe

Shiguo Wang 1, Na Li 1,

Wei Pan, Bo Tang*

College of Chemistry, Chemical

Engineering and Materials

Science, Engineering Research

Center of Pesticide and

Medicine Intermediate Clean

Production, Ministry of

Education, Key Laboratory of

Molecular and Nano Probes,

Shandong Normal University,

Jinan 250014, PR China

*Corresponding author.

E-mail: [email protected] authors contributed

equally.

0165-9936/$ - see front matter ª 2012

1. Introduction

Reactive oxygen species (ROS), reactivenitrogen species (RNS), reactive chloridespecies (RCS), reactive sulfur species (RSS),important cations, anions and amino acidsbelong to the family of intracellular small-molecule reactive species (ISMRS), whichare important small signaling molecules inphysiological and pathological cellularevents that govern cell functions [1–3].

The ROS family includes the superoxideanion radical (O2

��), the hydroxyl radical(�OH), hydrogen peroxide (H2O2), ozone(O3), singlet oxygen (1O2) and the peroxyradical (ROO�) [4]. ROS are mainly pro-duced by leaking of electrons from themitochondrial electron transport chain(ETC) [4]. By reduction of oxygen trans-ferred on ETC, NADPH catalyzes the pro-duction of a large variety of reactivemetabolites {e.g., the RCS family mediatedby myeloperoxidase (MPO)-H2O2-Cl� sys-tem and the RNS family mediated by NO}[5–7]. Overproduction and accumulationof oxidizing metabolites (e.g., ROS, RNSand RCS) could induce oxidizing or nitro-sative stress in living cells, which has been

Elsevier Ltd. All rights reserved. doi:http://dx.doi.org/10.1016/j.tr

reported to be harmful to the tissue [4–7].However, living cells have evolved elabo-rate mechanisms to maintain equilibriumof the reduction–oxidation (redox) state bycontrolling the precise balance betweenthe levels of oxidizing metabolites andreducing equivalents [e.g., ascorbate, thi-ols (RSH), ubiquinol, tocopherol andorganoselenium]. RSH are prominentmembers of RSS that control redoxhomeostasis mainly through the thiol-disulfide interchange reaction, which hasbeen implicated in many cellular pro-cesses (e.g., cell division and differentia-tion) [8].

To understand fully biological roles ofISMRS in living systems, it is crucial tomonitor accurately specific ISMRS at thecell, tissue and organism level. To this end,fluorescent and luminescent probes havebecome widely employed tools for real-time, non-invasive, sensitive and selectiveimaging of dynamic changes of ISMRS [9–12]. Conventional fluorescent probes withhigh signal-to-noise ratio, high quantumyield, membrane permeability and in vivostability could give selective response tospecific ISMRS and transduce changes of

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http://dx.doi.org/10.1016/j.trac.2012.07.010

Trends Trends in Analytical Chemistry, Vol. 39, 2012

ISMRS levels into a change in fluorescence intensity[13]. However, they merely display single-emission pat-terns or detect the overall levels of ISMRS without dis-tinguishing different concentrations of ISMRS fromdifferent organelles (e.g., mitochondria, nucleus, endo-plasmic reticulum and lysosome) when responding tooxidizing or nitrosative stress. Each organelle hasevolved a unique mechanism to deal with redoxhomeostasis, during which the function of each partic-ular metabolite of ISMRS may vary.

Emerging studies in molecular imaging suggest thatISMRS have a wide range of physiological effects on thecell network, depending on the specific identity of theISMRS, the concentration of the ISMRS, the timing andthe sub-cellular localization of their production [4,14–17]. Considering the complex biological environment,every particular metabolite of ISMRS needs to be moni-tored directly near the sites of production and action(in situ). Conventional fluorescent probes could not meetthe needs of researchers. Functional fluorescent andluminescent probes, including ratiometric fluorescentand luminescent probes, organelle-targetable fluorescentprobes, reversible fluorescent probes, multi-functional

Table 1. The progress of selected functional fluorescent and luminescentimaging in living cells

Year Milestone

1986 The first ratiometric and cell-trappable fluorescentprobe for Ca2+ that laid a solid foundation fordesigning ratiometric and cell-trappable fluorescentprobes

2002 The first ratiometric fluorescent probe for Zn2+

20062009

The first ratiometric fluorescent probe for H2O2

Increasing the sensitivity of cell-trappable fluorescenprobes by ester functionalization

2010 Cell-trappable probe for intracellular NO detectionthat brings the biochemistry of mobile Zn2+ and NObased on functional fluorescent probes to a new lan

201020102011

FRET-based fluorescent probe for �OH imagingThe first ratiometric fluorescent probe for Cu+

The first ratiometric fluorescent probes for NO andOCl�

2011 A series of H2O2 responsive probes that extendboronate oxidation as a prominent bioorthogonalreaction to design fluorescent probes

2011 Exploring the elaborate mechanism of mitochondriafunction and ROS

2011 Ratiometric fluorescent probes for Fe2+, ONOO� anCl�;reversible fluorescent probes for imaging redox cycle

2012 O3-responsive mitochondrial fluorescent probes2012 Ratiometric fluorescent probes for Cu2+; multi-

functional probe for simultaneously imaging H2O2

and NO to provide more precise data for detectingISMRS

ISMRS, Intracellular small-molecule reactive species.

4 http://www.elsevier.com/locate/trac

probes and corresponding nanoprobes substantially im-prove the performance of imaging probes and provide anew avenue to study ISMRS. For instance, organelle-targetable fluorescent probes could localize and accu-mulate within different organelles for a long time, soimproving on the sensitivity of traditional fluorescentprobes.

There is a growing interest in designing functionalprobes (Table 1) to study ISMRS at the sub-cellular level.Lippard brought the biochemistry of mobile Zn2+ and NObased on functional fluorescent probes to a new place[15]. Chang took the interplay between H2O2 and NOx astep further by employing functional small-moleculefluorescent probes based on a prominent bioorthogonalreaction (boronate oxidation) [16]. By utilizing target-able fluorescent probes, Murphy explored the elaboratemechanism of the mitochondrial function and ROS [17].Our group also developed a series of functional probes forbioimaging ISMRS [18,19].

In this article, we discuss ratiometric, reversible,organelle-targetable and multi-functional approaches indesigning probes so as to understand further the multiplebiological roles of ISMRS.

probes for Intracellular small-molecule reactive species (ISMRS)

Primary group leaders Ref.

Roger Y. Tsien [38,85]

K.R. Gee [39]

tChristopher J. ChangTetsuo Nagano

[52][92]

d

Stephen J. Lippard [15,93,94]

Bo TangChristopher J. ChangWeiying Lin

[51][41][55,59]

Christopher J. Chang [16,132]

l Michael P. Murphy [17]

d

s

Bo Tang [18,19,58][114]

Bo Tang [77]Weiying Lin [43,121]


2. Probes for ratiometric imaging of ISMRS

2.1. Design of ratiometric fluorescent and luminescentprobes for cell imagingConventional fluorescent probes for cell imaging convertintracellular ISMRS levels to switch on or off fluorescenceand display merely single-emission patterns after reactingwith target-specific ISMRS. However, the fluorescentproperties tend to be influenced by several factors undercomplex cellular conditions [e.g., the probe concentrationand changes in cellular microenvironments (e.g., tem-perature and pH)]. Ratiometric imaging is based on self-calibration of fluorescence intensity through measuringthe two distinct signals in the presence or absence ofISMRS, by which changes in concentration of the targetmolecule can be detected independent of such factors[20]. Thus, ratiometric imaging is a precise, practicalmeans for detecting ISMRS, as it allows for quantitativemeasurement of ISMRS levels and provides more accu-rate data, which is essential to address the challenges inelucidating the complex interplay between ISMRS andpathological events.

Most reported promising ratiometric systems are basedon ISMRS-induced modulation of fluorescence reso-nance-energy transfer (FRET), intramolecular chargetransfer (ICT) [20] between donor and acceptor, ornano-embedding {incorporation of reference dyes andspecific small-molecule fluorescent probes in a singlenanoparticle (NP) [21] (Fig. 1)}. Small-molecule dyes{e.g., cyanine [19], rhodamine [22], boron-dipyrro-methene (BODIPY) [23] and coumarin [24]} arefrequently employed as scaffolds for construction ofISMRS-responsive ratiometric probes. The frequentlyemployed fluorescent probe pairs for FRET are summa-rized in Table 2. These ratiometric systems have beenused to construct probes for imaging pH, anions, metalions and ROS/RCS/RNS/RSS [22–26].

Among them, FRET is a non-radiative energy-transferprocess, in which an excited dye donor transfers energyto a dye acceptor. In the presence or absence of ISMRS,imaging based on FRET allows the simultaneousrecording of two emission intensities at different wave-lengths. To design small-molecule FRET ratiometricfluorescent probes for ratiometric detection of specificISMRS, it is necessary to formulate a FRET platform,which comprises an energy donor, a linker and an en-ergy acceptor (Fig. 1). An appropriate interaction site forspecific ISMRS is then introduced on the FRET platformto modulate the energy-transfer efficiency [20].

For ICT, the most frequently used strategy is to modifythe electron-donating group (e.g., 4-amino donor) offluorophore into a more electron-withdrawing group(often carbamate with ISMRS-responsive moiety), sosuppressing an ICT process upon excitation by light canlead to a blue shift in its emission spectrum and result in

a ratiometric signal to allow quantitative determinationand imaging ISMRS [20,27,28]. The elaborately-de-signed ISMRS-responsive moiety within the carbamategroup could be specifically recognized by ISMRS and inresponse back to the amine, which would provide aswitch for ratiometric detection of the correspondingISMRS.

It is notable that, by incorporating different hydro-phobic dyes and specific small-molecule fluorescent probein a single NP without complicated synthetic andseparating procedures, fluorescent ratiometric nano-probes with good water dispersivity provide anotherfascinating and promising approach to constructratiometric probes in living cells [21]. Nanostructuredmaterials [e.g., polymers, fluorescent silica NPs and up-converting luminescent NPs (UCNPs)] are frequently em-ployed for ratiometric dual-wavelength fluorescent andphotoluminescent imaging [29–35]. In this review, we donot detail fluorescent protein probes that require geneticmanipulation, which frequently adds complications [36].

2.2. Ratiometric fluorescent probes for cationsThe essential activity of cations is indispensable to cel-lular events [9,37–47] (e.g., Fe3+/Fe2+ and Cu2+/Cu+

are reported increasingly to be involved in the metabo-lism of ROS and RNS). Fe2+ and Zn2+ may also facilitatethe generation of ROS or RNS, respectively. Ca2+, Na+

and K+ are the essential ions that maintain charge bal-ance and electron transfer. These metal ions are keymodulators of biochemical-signaling pathways in neu-rons, so there is a growing interest in designing ratio-metric probes for metals, which are receiving greaterattention to bridge the gap.

More than a decade ago, Tsien et al. designed the firstratiometric Ca2+-sensitive probe 1a based on the Fura-2family, which gave selective response to Ca2+ over othermetal ions [38]. Ten years ago, Gee et al. developed thefirst ratiometric Zn2+-sensitive probe 1b based on ex-cited-state intramolecular proton transfer (ESIPT) tovisualize the movement of Zn2+ in cells [39].

Since that time, several metal ion-responsive ratio-metric fluorescent probes have been developed [16](Fig. 2). These probe designs are generally based on FRET,ICT, intracellular ester hydrolysis and nano-embedding.The interesting photochemical properties of selectedradiometric fluorescent probes are summarized inTable 3. However, some probes based on the Fura-2 andIndo-1 family require UV excitation, which can be dam-aging to cells and present problems with autofluorescencewhen applied in vitro. Among them, the cell-permeableZn2+ ratiometric fluorescent probe 2 designed by Lippardet al. requires close attention [40]. In probe 2, the ratio-metric imaging of Zn2+ was based on Zn2+-controlledswitching between fluorescein and naphthofluoresceintautomeric forms with single-excitation, dual-emission

http://www.elsevier.com/locate/trac 5

(a)

ISMRS

FRET or ICT

Ex Em1

Ex Em2

(b)

ISMRS indicator

ISMRS indicator

Reference dye

Figure 1. Mechanism of ratiometric fluorescent probes (a) FRET or ICT, (b) embedding or surface-functionalization of nanoparticles.

Table 2. Fluorescent probe pairs for FRET

Ref. Donor(D) Wavelength(nm) Acceptor(A) Wavelength(nm) Comments

[24,47] Cy3Ex:550, Em: 570

Cy5Ex: 650, Em: 670

Cyanine: easy-modification and emission in NIR region inwhich background autofluorescence can be eliminated

[24] BODIPYEx485, Em:510

RhodamineEx: 550, Em: 589

BODIPY: high extinction coefficient, high quantum yields,excitation and emission profiles insensitive to solvent polarityand pH

[43,55] CoumarinEx: 410, Em: 473

RhodamineEx:560,Em:583

Rhodamine: high absorption coefficient, high fluorescencequantum yield, photostability and broad fluorescence in thevisible region

[51] FAMEx: 496, Em: 518

AuNP(quencher)

Fluorescein dyes: excellent labeling fluorophore, highquantum yields and water solubility;

[52] CoumarinEx: 420, Em: 464

FluoresceinEx:461,Em:517

Coumarin: large Stokes shift, easily conjugated to other dyesfor efficient energy transfer

[63] CoumarinEx: 420, Em: 459

PorphyrinEx:421, Em:658

Porphyrin: tunable photophysical properties by modificationson the nucleophilic hydroxyl groups

The term ‘‘cyanine dye’’ denotes a dye system with a polymethine chain between two nitrogens (i.e. R2N-(CH‚CH)n-CH‚N+R2). Cyanine dyesare given common names according to the number of carbon atoms between the dihydroindole units. Cy3: cyanine dye with three carbon atomsbetween the dihydroindole units; Cy5: cyanine dye with five carbon atoms between the dihydroindole units; BODIPY: boron-dipyrromethene;FAM: carboxyfluorescein; AuNPs: gold nanoparticles


profiles. Seminaphthofluorescein was chosen as the fluo-rophore and 2-[bis(2-pyridylmethyl)-aminomethyl]ani-line as the receptor of Zn2+. Probe 2 showed excellentselectivity responses for Zn2+ over competing Ca2+ and


Mg2+ at intracellular concentrations with dissociationconstant (Kd) for Zn2+ of <1 nM and an 18-fold increasein fluorescence-emission intensity ratio (F624/F528) uponzinc binding. It could detect endogenous stores of intra-

Table 3. Photochemical properties of ratiometric fluorescent probes

Probes Wavelength shifts Comments Ref.

1a The binding of Ca2+ shifted the fluorescence excitationspectrum of probe to shorter wavelengths at 340–350 nm witha depressed excitation spectrum at 380–390 nm. The emissionspectrum peaks at 505–510 nm and hardly shifts wavelength

The first ratiometric fluorescent probe for Ca2+ with a selectiveresponse to Ca2+ over other metal ions

[38]

1b Fluorescence wavelength shift from 480 nm to 395 nm(excitation at 350 nm)

The Zn2+ dissociation constant of the probe is 3.0 lM. showinga high affinity for Zn2+

[39]

3 Fluorescence wavelength shift from 570 nm to 556 nm withthe emission intensity at 505 nm unchanged (excitation at480 nm)

It showed 20-fold fluorescence ratio change upon Cu+ binding [41]

6 The fluorescence intensity at 635 nm was quenched with theemission intensity at 507 nm unchanged (excitation at 485 nmand 569 nm)

Near-infrared fluorescence could reduce the interferencesfrom background fluorescence and light scattering

[19]

8 With decreasing pH, the ratio of fluorescence intensitiesemitted from donor and acceptor dyes at two emissionwavelengths, 570 nm and 670 nm increased (excitation at543 nm)

It could discriminate variations in acidity of cellular organelles [47]

10 The referenced signal 670 nmn800 nm increases up to 4.5-foldfrom oxygen-saturated to oxygen-free conditions (excitation at635 nm)

The nanosensors could be targeted for imaging of hypoxicconditions due to accumulation of the nanosensors in tumortissue

[48]

11 The fluorescence intensity ratios of probe at 495 nm and651 nm showed a dramatic change from 0.018 to 3.8 upontreatment with �OH (excitation at 460 nm)

The probe features high selectivity for �OH, a very largeemission shift (156 nm), a large ratiometric signal (a 210-foldvariation), emission in the NIR, high stability in solution,excitation in visible light, cell-membrane permeability

[50]

14 The ratio of emission intensities (F540/F475) varies from 0.3 to3.6 after treatment with H2O2 (excitation at 410 nm)

The observed rate constant for H2O2 deprotection,kobs = 8.8·10�4/s. The ratiometric emission response of theprobe is highly selective for H2O2 over other reactive oxygenspecies

[36]

17 The novel ratiometric fluorescent sensor exhibited a very largevariation (up to 420-fold) in the fluorescence ratio (F583/F473)(excitation at 410 nm)

The probe features high sensitivity, high specificity,functioning well at physiological pH, low cytotoxicity, andgood cell-membrane permeability

[55]

18 The nanoprobe gave different emission wavelengthsF575/F810 (excitation at 532 nm and 635 nm, respectively)

The probe has the unique advantages of good water solubility,photostability, biocompatibility, andNIR excitation and emission

[18]

20 The fluorescence intensity at 436 nm dropped gradually,excited at 318 nm and fluorescence intensity unchanged at519 nm (excited at 494 nm with increased Cl� concentration)

The probe is stable with an instantaneous response tofluctuation of Cl� concentration. It realized the dynamic,visualizable analysis of Cl�-level differences between normaland ischemic ventricular myocytes

[58]

21 It displayed an 80 nm red shift (excitation at 540 nm) The probe exhibited high sensitivityand selectivity to OCl�

[59]

23 The emission ratio F459/F658 showed a dramatic increase in thepresence of increasing concentrations of Cys (excitation at350 nm)

It is highly sensitive to thiols with a low detection limit and alarge fluorescence dynamic range

[63]

ISMRS: Intracellular small-molecule reactive species


cellular Zn2+ after NO-induced release of Zn2+ from cel-lular metalloproteins. This connection of Zn2+ and NOlaid a solid foundation for studies of the connection be-tween Zn2+ and RNS.

Cu2+/Cu+ are involved in bone formation, cellularrespiration and connective tissue development. Shiftingthe Cu2+/Cu+ redox equilibrium is closely connectedwith oxidative stress and neurological disorders,including Alzheimer�s, Parkinson�s, Menkes� andWilson�s diseases [41]. Thus, ratiometric detection ofCu2+/Cu+ redox equilibrium is of paramount signifi-cance. So far, for specific ratiometric imaging intracel-lular Cu2+/Cu+ homeostasis, ratiometric fluorescentprobes 3, 4 and 5 have been developed. In 2010, Chang

et al. developed ratiometric Cu+ fluorescent probe 3composed of an asymmetric BODIPY as fluorophore andthioether-rich receptor as modulator for binding Cu+

[41]. In absence or presence of Cu+, the emissionintensity at 505 nm was unchanged but the emissionband at 570 nm changed dramatically, which made 3suitable for ratiometric imaging of intracellular Cu+

homeostasis. Probe 3 showed high selectivity for Cu+

over other metal ions and a �20-fold fluorescence ratiochange with visible excitation and emission profiles.

In 2011, Lu et al. reported the importance of dual-emission fluorescent silica NP-based Cu2+ probe 4 thatcomprised a dye-doped silica core as a reference signaland a Cu2+ receptor covalently grafted on the surface of


O

N

O

COO-

OCH2CH2O

N(CH2COO-)2 N(CH2COO-)2

CH3

1a

NH

CO2K

OCH3

N

CO2K CO2K

1b

O

HN

N

N

N

OH

CO2H

O

2

NB

N

F F N

SS

SS

H3CO

3

4

O

N N

N

O

N

N N

O

OO

NH2

O

5

Figure 2. Structures of ratiometric fluorescent probes 1–8 for imaging cations.

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N

N

N

HN O

NB

N

NN+

I-

SO3K

KO3S

FF

6

NB

N

FF

7COOCH3

N

N H

Figu

re2.

(conti

nued

)



N

HN O

NO2

O

O O

CH2CH2OCH2CH2OOCCH3

9

10

ON O

H4'

N

11

12

Figure 3. Structures of ROS-activated ratiometric fluorescent probes 9–16.

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B

O

O

O

O

O

NH

HN

O

O

O

O(H3CH3C)2N

N

HN

O

BO

O

O

OO

CH2CH2OCH2CH2OOCCH3

14

B

O

O

15

O

HN

O

O

B

OO

16

NO

O S

N

HN

PPh3+

Figure 3. (continued)

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O

NN

O

ON O

O

N

O

O

N N

H2N

17

18

N

N N

N N

CO2-

CO2- -O2C

-O2C

OCH3

OCH3

Eu3+ or Tb3+

19

Figure 4. Structures of ratiometric fluorescent and luminescent probes 17–19 for sensing RNS.

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N

(H2C)2

C

HN

O

H3CO

+

O

CO2H

O

20

ON O

NHN

O2N

NO2

21

Figure 5. Structures and confocal imaging of fluorescent probes 20 and 21 for sensing RCS in living cells. Ratiometric imaging of Cl� in ischemic ventricular myocytes (left) and inOCl� in live MCF-7 cells (right) incubated with probes.

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ON O

22

CHO

23

N

HNNH

N

OSS

O

NHO

O

HO

OH

HO

OH

O

24

NO

O S

N

HN

PPh3

O

+

S

SNH3+

Figure 6. Structures of RSH-responsive ratiometric fluorescent probes 22–24.

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Non-fluorescent indicator

Fluorescent indicator

Figure 7. Mechanism of organelle-localized ISMRS fluorescent probes.

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15

NH

NH

N

NN

HN

O

N-O2C

CO2-

O O

N CO2-

CO2-

N

N NH

N

OO

H2N

25

Figure 8. Structures of organelle-localized ISMRS fluorescent probes 25–27 based on SNAP tag.

26

N

NN

NH

O

HN

CO2H

OOH

Cl Cl

NH2

O

O

N N

NN NN






the silica NPs. Nanoprobe 4 exhibited a ratiometricfluorescence response to Cu2+ in HeLa cells with limit ofdetection (LOD) as low as 10 nM [42].

In 2012, ratiometric Cu2+ fluorescent probe 5 wasdesigned on the basis of coumarin-rhodamine scaffold byLin et al. [43]. FRET is one of the commonly exploitedsensing mechanisms for design of ratiometric fluorescentprobes. Modulating ratiometric fluorescence of 5 wasachieved by FRET from energy donor (coumarin) toacceptor (rhodamine), which led to a large emission-wavelength shift (around 110 nm) between coumarinemission and rhodamine emission. The ratiometricfluorescent probe featured a low LOD (S/N = 3) of 13 nMin HEPES buffer.

Cyanine has found many biological applications indesigning probes because of its easy modification andemission in the near-infrared (NIR) region (650–900 nm), in which interference from background auto-fluorescence by intracellular biomolecules can be elimi-nated [44,45]. However, it is still rare to report

constructing ratiometric fluorescent probes based oncyanine.

For ratiometric detecting Fe2+, our group prepared anew NIR fluorescent probe 6 based on cyanine byintroducing BODIPY to replacing hydroxyl as the refer-ence fluorophore and the group 4 0-(aminomethylphe-nyl)-2,2 0:6 0,200-terpyridine (Tpy) as selectively responsiveto Fe2+. This method could be extended to construct otherNIR ratiometric fluorescent probes [19]. Ratiometricfluorescent probe 6 showed good selectivity and highsensitivity with the LOD as low as 12 nM upon bindingwith Fe2+. The probe was stable and could instanta-neously respond to Fe2+-concentration fluctuations withgood cell-membrane penetrability and low toxicity.

As minor variations of intracellular pH may inducecellular dysfunction, desirable pH fluorescent probesshould be able to respond sensitively to minor changes ofpH, and to avoid interference from native cellular spe-cies. Imaging and probing the intracellular acidicorganelles (e.g., early/late endosomes and lysosomes)



could provide valuable information about disease local-ization and offer a practical tool for medical therapy anddiagnostics. Ratiometric fluorescent probes could probeeither molecular interactions or a single molecular event(e.g., detecting acidation in molecular environments).Ratiometric pH probes 7, based on the photoinducedelectron-transfer (PET) mechanism, and 8, based on theFRET mechanism, were developed for detecting/imagingintracellular pH changes by Alvarez-Pez et al. [46] andSung et al. [47], respectively. Ratiometric pH probe 7was prepared by conjugatively linking the BODIPYfluorophore at the 3-position to pH-sensitive ligandimidazole through an ethenyl bridge [46]. PET occurredefficiently under neutral conditions but inefficiently un-der acidic conditions. The modest pKa value of 4.9 issuitable for detecting changes in the acidic environmentin living organisms.

Ratiometric pH nanoprobe 8 was developed based onN-palmitoyl chitosan bearing a donor (Cy3) and anacceptor (Cy5) moiety [47]. At low pH, changes in theenvironmental pH enabled 8 to gain FRET and moreenergy transferred from Cy3 to Cy5.

Both 7 and 8 showed the pH-dependent spectralproperties that exactly detected H+ and afforded moreprecise data for analysis of roles of H+ in ROS metabolism.

2.3. Ratiometric fluorescent and luminescent probes forROSIn the metabolism of mitochondria, an inadequate oxy-gen supply could induce hypoxia, which is a feature ofmany pathological processes (e.g., cancers). Hypoxia isassociated with increased metabolic activity and resultsin an elevated extracellular acidity, so detection andratiometric imaging of oxygen and hypoxia in solid tu-mors is of paramount clinical significance for under-standing the biological role of oxygen [27,48].Intracellular fluctuation of local oxygen concentrationscan be exactly measured by ratiometric fluorescenceintensity.

Recently, for detecting hypoxia, ratiometric fluorescentprobe 9 [27] and nanoprobe 10 [48] were developed byQian et al. and Schaferling et al., respectively (Fig. 3).Probe 9 was developed by connecting a p-nitrobenzylmoiety and signaling moiety 4-amino-N-(2-(2-acetoxy-ethoxy) ethyl) naphthalimide (RHF) via a carbamategroup. Upon reduction by oxygen-sensitive nitroreduc-tase (NTR), the amino group of RHF was released togenerate the aminonaphthalimide and ICT occurred,resulting in the restoration of fluorescence emission. Themaximum emission wavelength of 9 was at 475 nm whileRHF was longer by �75 nm and fell at 550 nm, whichmade probe 9 favorable for ratiometric imaging of hy-poxia.

Luminescent NIR polymer-nanoprobe 10 was devel-oped for in vivo oxygen sensing [48]. Nanoprobe 10 was


based on polystyrene NPs (PS-NPs) incorporating anoxygen-sensitive NIR-emissive palladium meso-tetra-phenylporphyrin and an inert reference dye, which wereboth excitable at 635 nm.

Generated by successive monovalent reduction of O2

in the course of biological metabolism, �OH is a veryreactive species that has a lifetime of about 2 ns inaqueous solution [49]. The concentration of �OH is lowin biological systems. Their fluorescence signals sufferfrom interference by other highly reactive oxygen species(hROS0, which are strong oxidants that include �OH,hypochlorous acid (HOCl) and peroxynitrite (ONOO�). Inparticular, interference from ONOO� is a severe obstaclein detecting �OH because of the similarity betweenONOO� and �OH with regard to their strong oxidationcapacity.

Ratiometric �OH fluorescent probes 11 and 12 weredeveloped by Lin et al. [50] and our group [51],respectively. To find applicability in living cells, Linprepared ratiometric fluorescent probe 11 for �OHratiometric imaging in 2010. Probe 11 was prepared byhybrid coumarin–cyanine dye [50]. In the absence of�OH, the free probe displayed an emission band centeredat 495 nm. However, upon treatment with �OH, therewas a marked decrease of emission intensities at 495 nmand concurrently a significant enhancement of emissionintensities at 651 nm.

Ratiometric �OH fluorescent probe 12 comprised car-boxyfluorescein (FAM) as fluorophore and gold NPs(AuNPs) as quencher modules linked by single-strandedDNAs. Based on the hybrid FAM-DNA-AuNP platform,the fluorescence of FAM was quenched. However, �OH-induced cleavage of DNA strands would release the freeFAM with strong fluorescence [51]. This methodologyestablished the value of this nanotechnology-based probefor imaging �OH in living cells and opened a new windowto facilitate investigations of ROS-mediated cell behavior.

Chang et al. fully explored an H2O2-activatable ratio-metric fluorescent probe and devised 13–15 based onboronate oxidation [28,52,53]. Ratiometric probe 13was designed based on FRET, and comprised a coumarindonor and a boronate-protected fluorescein acceptorlinked by a rigid spacer [52]. In the absence of H2O2, as aresult of suppressed FRET, only blue-donor emission wasobserved upon excitation of the coumarin chromophore.The FRET-based probe showed a >5-fold higher emis-sion-ratio response to H2O2 over other competing ROS(e.g., O2

�� or tert-butyl hydroperoxide). Though 13 wassuccessfully applied for selective detection of H2O2 inseparate mitochondria and capable of quantifyingchanges in H2O2 through a ratiometric fluorescence re-sponse, it was not extended to cell imaging. The ratio-metric imaging probe 14 was extended to live tissue bytuning the ICT properties through modulating the elec-tron-donating 4-amino donor on 1,8-naphthalimide,

NH N

N

N

N

S

N

HN

O

Ph3P+

28

Ph3P+

NH

N

NO O

O O

O

O

NH

O

N N

O

O

O

29

Figure 9. Structures of mitochondria-targeted fluorescent probes 28–35 for detecting ISMRS.

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30

HN

PPh 3

+

O

O

NB

N

N S

S

S

S

H3CO

FF

Ph 3P+

31

S

N HN N

O

O.

OHO

OH

O

Figu

re9.

(conti

nued

)



BO

O

O

O

32

ON

NPh3P+

HN CO2H

NN

N

NH

+I-

33

HN CO2H

NN

HN

+I-

34

O

O

NH2

N N+

Cl-

35a

O

O

OH

N N+

Cl-

35b


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which affected both ICT and emission color, as makingthis substituent more electron-deficient resulted in ICT-induced blue shifts in emission maxima [28].

In 2011, two-photon probes 15 [53] and 16 [54] forH2O2 were developed for deep tissue imaging. Both 15and 16 could ratiometrically detect H2O2 in live cells andintact tissues at > 100 lm depth through the use of two-photon microscopy (TPM). 16, especially, could monitorchanges in mitochondrial H2O2, as reported by Kim et al.[54]. TPM provides imaging with increased depth ofpenetration (4500 mm) and prolonged observation time,due to the use of two NIR photons as the excitationsource with less-damaging lower energy light [54].

2.4. Ratiometric fluorescent and luminescent probes forRNSNitric oxide (NO) is a diffusible gaseous free radical withan average life-time of ms–s and a large range of bio-

Figure 10. Structures and confocal imaging of fluorescent probes 36 and 3rescence lifetime image (upper middle) of fluorescent probe 36 showing aboth HEK293 and HeLa cells loaded with fluorescent probe 37 and the nucestablishing that fluorescent probe 37 accumulates specifically in the nucl


logical concentrations (nM–lM), so it tends to formderivatives rapidly in physiological conditions. RNS playa multi-functional role in both physiological and path-ological processes [7].

In 2011, Lin et al. developed ratiometric fluorescentNO probe 17 (Fig. 4), which comprised a coumarin do-nor, a rhodamine acceptor, and a rigid piperazine linker,by taking advantage of the ring-opening process of therhodamine spirolactam induced by NO, which switcheson FRET and brings variations in optical properties [55].However, in the absence of NO, the rhodamine acceptorwas in the closed form, which meant the excitationenergy of the coumarin donor could not be transferred tothe rhodamine acceptor. Ratiometric fluorescent probe17 exhibited a highly sensitive ‘‘turn-on’’ fluorescentresponse toward endogenously-produced NO inmacrophage cells. Remarkably, there is a �420-foldvariation in the fluorescence ratio (I583/I473) from 0.061

7 in living cells. Confocal fluorescence image (upper left) and fluo-higher temperature in the nucleus (upper right). Confocal imaging oflear stain Hoechst 33342 shows good colocalization of the two dyes,eus (lower).

B

O

OO

O

O

NH

37



in the absence of NO to 26.6 in the presence of 100equiv. NO.

To solve the selectivity of NIR fluorescent probes to-wards ONOO� and to monitor accurate fluxes of ONOO�,our group developed 18 for highly selectively monitoringthe concentration changes of intracellular ONOO� bymeasuring the ratio of red and green fluorescenceintensities [18]. Nanoprobe 18 was based on a polymericmicelle made of poly(d,l-lactic acid) (PLA) and polyeth-ylene glycol (PEG) by incorporating it into rhodamine asreference dye and benzylselenide-tricarbocyanine (BzSe-Cy) as ONOO� indicator, which gave selective response toONOO�. It effectively avoided the influences from enzy-matic reaction and high-concentration �OH and OCl�.

Ratiometric luminescence probes 19 for detecting andimaging ONOO� in HeLa cells was developed by Yuanet al. [56]. [Eu3+/Tb3+ (DTTA)] mixture was employed asa ratiometric probe for detecting ONOO�. The lumines-cence of [Tb3+ (DTTA)] could be quenched by ONOO�

rapidly while [Eu3+ (DTTA)] did not respond to ONOO�.Luminescent lanthanide complexes have long lumines-cence lifetimes, large Stokes shifts, and sharp emissionprofiles; these properties enable them to be used for lstime-gating. However, ratiometric luminescence probeshave absorption in the UV region, which renders themunsuitable for imaging ISMRS.

2.5. Ratiometric fluorescent probes for RCSRCS mainly include HOCl and Cl�. HOCl is a highlyreactive species produced from peroxidation of Cl� cat-alyzed by the enzyme MPO. Mismanagement of them hasa close relationship with cardiovascular diseases, myo-tonia and cystic fibrosis [6,57–59]. Cl� concentration inventricular myocytes is clinically regarded as a feature ofmyocardial ischemia, so there is currently great interestin developing ratiometric fluorescent probes that canselectively detect intracellular HOCl and Cl�.

In 2011, our group developed ratiometric Cl� fluo-rescent probe 20 (Fig. 5). 5-Amino-fluorescein (AF) was

chosen as reference dye and 6-methoxyquinoline(MQMBP) as Cl� recognition receptor [58]. When theprobe was excited using the wavelength of 318 nm(exciting the MQMBP moiety), the fluorescence intensityat 436 nm dropped gradually with the increase of Cl�

concentration. Meanwhile, the fluorescence intensity ofthe AF moiety, as the Cl�-insensitive group around519 nm, kept nearly constant when the probe was ex-cited at 494 nm. Thus, the fluorescence intensity of 20decreased linearly with the increase of Cl� concentra-tion. Probe 20 showed excellent linearity between fluo-rescence ratios (F519/F436) and Cl� concentrations in therange 0.5–100 mM, which correlated well with the Cl�

level in ventricular myocytes. The LOD was calculated tobe 0.48 mM. These results showed this probe couldqualitatively and quantitatively detect Cl� in ventricularmyocytes.

For selective detection and imaging of intracellularHOCl, ratiometric fluorescent probe 21 was designed byLin et al. [59] by taking advantage of the transformationof coumarin-2,4-dinitrophenylhydrazone to coumarinaldehyde promoted by OCl�. The intensity of the emissionpeak of 21 at 585 nm gradually decreased, while the newblue-shifted emission peak centered at 505 nm increased,so 21 could give a ratiometric response to OCl� and wassuitable for imaging OCl� in live MCF-7 cells.

As the levels of HOCl are closely connected withhomeostasis of pH and ROS, ratiometric fluorescentprobes and multi-functional fluorescent probes capableof simultaneously monitoring endogenously-producedROS and RCS are greatly needed for further studies.

2.6. Ratiometric fluorescent probes for RSSRSH are important indicators and modulators in intra-cellular redox homeostasis and cellular growth involvedin human health and disease [5,8,60–64]. More pre-cisely, in mitochondria, small-molecular-weight thiols[e.g., cysteine (Cys), homocysteine (Hcy), and glutathione(GSH)] constitute the RSS family, which includes thiols,


N

NN+

I-

38

HN NHOH HO

OO

NB

N

NR2R1

OHHOF F

OO

39a R1, R2= H

39b R1, R2=CH3

39c R1=CH3, R2=CH2CH3

39d R1, R2=CH2CH3

NB

N

NR4R3

R1

F F

R1

R2 R2

40a R1, R2, R3, R4= CH3

40b R1, R2=CH3, R3, R4=CH2CH3

40c R1, R2=CH3, R3=CH2CH3

R4=CH2CH2CH2CO2CH3

40d R1=H, R2=CH2CH2CO2CH3

R3, R4=CH2CH3

Figure 11. Structures of fluorescent probe 38–41.

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S-nitrosothiols, sulfenic acids, and sulfite. They controlredox homeostasis through the thiol-disulfide inter-change reaction, which has been implicated in manycellular processes (e.g., cell division and differentiation)[8].

Lin et al. reported ratiometric fluorescent probes 22and 23 for specific detection of RSH [62,63] (Fig. 6).Probe 22 could detect Cys over Hcy and GSH based oncoumarin aldehyde [62]. In the absence of Cys, probe 22displayed an emission band at 557 nm, while, upon

N(CH2COOCH2OCOCH3)2

OCH2CH2O

CH3

N(CH2COOCH2OCOCH3)2

42 R=

Figure 12. Structures of cell-trappa

O OO

OORO

N

O

CO2HCO2H

N

R=

44

HO

Cl

O

CO2COCH3

45b

H3COOC

Cl

OCOCH3

Cl

ClH2C

H

Figure 12. (co

treatment with Cys, it showed dramatic variation. Therewas a marked decrease of emission intensity at 557 nmand concurrently a dramatic enhancement of emissionintensity at 487 nm, which made 22 suitable for imag-ing Cys in living cells.

Probe 23 was constructed for detecting intracellularRSH based on FRET from coumarin to porphyrin, as thecoumarin emission spectra matched well with the por-phyrin absorption spectra [63]. The addition of Cyselicited significant fluorescence enhancement at 459 nm

O

CO2H

HN NH

N N

OR RO

O

O

O

43

HO

ble fluorescent probes 42–50.

O

CO2COCH3

45a

OH

Cl

ClH2C

H

O

O

B O O

OO

O

O

O

O

O

O

46

ntinued)


O

O

B N

O

O

O

47

N

O

O

O

O OHO

NH2

NH2

48

N N

HO2C

CO2H

CO2H

HO2C

ClCl

CO2H


O OO

49

N N

HO2C

CO2H

CO2H

HO2C

CO2H

NH2



(emission wavelength of coumarin) but almost nochanges in fluorescence intensity at 658 nm (emissionwavelength of porphyrin). The emission ratio F459/F658

showed a 60-fold enhancement from 0.21 in the absenceof Cys to 12.1 in the presence of Cys (500 equiv). Theemission ratios F459/F658 exhibited excellent linearitywith Cys in the range 1–600 lM. The LOD (S/N = 3) ofthe probe was determined to be 0.73 lM. The combi-nation of the low LOD and the large fluorescence dy-namic range indicated that it was suitable for ratiometricimaging and detecting RSH in HeLa cells.

Kim et al. reported a ratiometric two-photon fluores-cent probe 24 for selective detection of mitochondrialthiols [64]. A disulfide group was selected as thethiol-reaction site, 6-(benzo[d]thiazol-2 0-yl)-2-(N,N-


dimethylamino) naphthalene (BTDAN) as the reporterand triphenylphosphonium salt (TPP) as the mitochon-dria-targeting site.

2.7. Challenges for ratiometric fluorescent probesBased on self-calibration of fluorescence intensity toerase the potential origin inaccuracy of cell imaging,ratiometric fluorescent probes have been widely appliedfor imaging ISMRS [22–26], which is a majoradvancement in developing fluorescent probes. However,there are still several challenges in the development ofratiometric fluorescent probes:

(1). the sensitivity of ratiometric fluorescentprobes in detecting ISMRS needs to be muchhigher;

O

CO2H

O

50a

O

Cl

NH

CuII

N

Cl

O

OO


O

CO2H

O

50b

O

Cl

NH

CuII

N

Cl

O

NH

dextran

O



(2). the species (e.g., Fe2+, Cu2+, Cl�, O2, ONOO�)that frequently cause quenching of fluorophoresupon collision with the fluorescent probes areintractable to be detected by conventional fluo-

rescent probes, and ratiometric fluorescentprobes designed for detecting these species willbe more necessary.



Future research on detecting ISMRS will benefit fromthese ratiometric fluorescent probes.

3. Organelle-localized ISMRS fluorescent probes

Genetically-encoded tags (e.g., GFP) are traditionallyused to monitor ISMRS in situ. However, they can causesignificant perturbations to protein structure, whichwould have an effect on imaging ISMRS [36]. Comparedwith this technique, SNAP-tag (SNAP-tag is derived fromthe 20 kDa DNA repair protein O6-alkylguanine-DNAalkyltransferase (AGT) and is labeled using O6-benzyl-guanine derivatives)-containing fluorescent probes andorganelle-targetable small-molecule fluorescent probesthat operate more conveniently offer an alternativemethodology for designing organelle-localized probesand studying localized ISMRS fluxes [12,17,65–84].SNAP-tag employs transfection using standard liposome-mediated gene-transfer techniques [12,65,67–69].Organelle-targetable fluorescent probes can be achievedby incorporating or covalently modifying a unique bio-orthogonal chemical tag into a conventional fluorescentand luminescent probe. The bioorthogonal chemical tagis often as small as a single functional group that couldaccumulate in specific organelles (Fig. 7). For the visu-alization of local ISMRS signaling in specific organelles,several bioorthogonal chemical tags that could beincorporated into single fluorescent probes have beendeveloped [17,66–84]. Recently, organelle-localizedprobes for ISMRS became very popular, some beingparticularly useful to assess homeostasis of thiols [64],Zn2+ [67,70,71], Ca2+ [68], Cu+ [72,81], H2O2

[69,74,80], 1O2 [76], O3 [77], hROS [78] fluxes, oxida-tive stress [73], temperature changes [79] and pH fluc-tuations [82–84] in different organelles.

3.1. Strategies to design and to fabricate sub-cellulartargetable fluorescent probesISMRS-responsive organelle-targeted probes couldbe selectively targeted to nucleus, mitochondria,endoplasmic reticulum and plasma membrane by fusionof SNAP-tag to the C-terminus of the histone H2Bprotein (SNAP-H2B), the C-terminus of cytochrome coxidase subunit 8 (Cox8A), a signaling peptide forretention of the protein in endoplasmic reticulum(KDEL), the N-terminus of neurokinin-1 receptor (NK1R)and the C-terminus of 5HT3A serotonin-receptor-signaling sequence.

In 2008, Lippard et al. designed organelle-specificZn2+ probe 25 that combined the Zn2+-sensitive sensorand AGT substrate benzylguanine [67] (Fig. 8). By fu-sion with human-1, 4-galactosyltransferase to directAGT to the trans-Golgi cisternae and sub-unit VIII ofcytochrome c oxidase to direct AGT to the mitochondrial


matrix, these probes could separately monitor Zn2+ fluxin Golgi apparatus and the mitochondria.

In 2009, Rıos and Johnson et al. introduced a similarmethod to measure Ca2+ concentrations inside nuclei ofliving cells, based on linking Ca2+-sensitive dye 26 Indo-1 to SNAP-tag fusion proteins [68].

In 2010, Chang et al. exploited the versatility of theSNAP-tag technology for site-specific protein labelingwith the use of boronate-capped dyes 27 to visualizeH2O2 selectively in different organelles [69]. These H2O2-responsive organelle-targeted reporters were selectivelytargeted to nucleus, mitochondria, endoplasmic reticu-lum and plasma membrane.

3.2. Mitochondria-targetable fluorescent probesMitochondria is a conventional primary source of ROS inthe course of metabolizing oxygen and releasing surplusenergy in the form of heat through respiration. On theETC in mitochondria, NADPH catalyzes the productionof large variety of reactive oxidants, including ROS, RCSand free radicals. High levels of ROS can trigger celldeath, whereas lower levels drive diverse and importantcellular functions. To distinguish the variable concen-tration of ROS in mitochondria will be of paramountsignificance. When there is higher Zn2+ concentrationsin living cells, O2

�� production increases, which resultsin mitochondrial dysfunction, formation of additionalONOO�, and amplification of the Zn2+ and NO signalingpathway, which lead to cell death. The fluorescent probetargeting the intracellular mitochondria is through theuse of small chemical tags bearing lipophilic cationsthat are attracted to the inner mitochondrial membranewith negative potential. So far, such small chemical tagsare:

(1). TPP tag;(2). mitochondria-targeted peptides (MPPs); and,(3). environmentally sensitive fluorophores that can

be specifically targeted to mitochondrial micro-environments (e.g., MitoTracker, rhodamineand cyanine dyes) [17,65,75].

The fluorescent probes incorporated with smallchemical tags bearing lipophilic cations possess anoverall positive charge, so they could accumulate in themitochondria of living cells for a long time. Confocalmicroscopy experiments further validate that thetargetable fluorescent probes accumulate within themitochondria of living cells.

For detecting mitochondrial Zn2+ fluxes, 28 and 29have been developed [70,71] (Fig. 9). In 2011, Kim et al.reported two-photon probe 28 for mitochondrial Zn2+

[70]. The probe showed a 7-fold enhancement of two-photon-excited fluorescence in response to Zn2+ withoutinterference from other metal ions, allowing the detec-tion of Zn2+ in a rat hippocampal slice at a depth of100_200 lm through the use of TPM.


In 2011, Davidson and Zhu et al. developed a mito-chondria-localizing Zn2+ probe 29 by introducing a TPPmoiety to the side of diamino-substituted naphthalene-diimide [71]. Fluorescent imaging of 29 in living TPMcells showed its good colocalization with mitochondria,which made it suitable for selectively detecting Zn2+ overother mitochondrial metal ions (e.g., Fe2+ and Cu2+/Cu+).

In 2011, Chang developed mitochondria-targetedfluorescent probe 30 for monitoring Cu+ homeostasis[72]. Probe 30 was achieved by introducing a carbox-ylic-acid handle conjugated with a TPP tag into probe 3.Using probe 30 revealed that Cu+-deficient SCO1 andSCO2 (two syntheses of cytochrome c oxidase genes)patient cells prioritize mitochondrial copper homeostasis.

In 2007, Miyata et al. developed TEMPO derivative 31that has a nitroxyl-radical moiety for measuring ESR,and fluorescein moiety for confirming distribution incells with TPP moiety for evaluating oxidative stress inmitochondria [73].

In 2008, Chang et al. employed newly-developedmitochondria-targeted H2O2 probe 32 by introducingthe H2O2–cleavable boronate switch and a TPP tag intothe rhodamine scaffold [74]. Probe 32 featured a turn-on response for H2O2 and could selectively detect sub-cellular changes in H2O2 fluxes in the mitochondria ofliving cells.

Permeant cationic fluorescent probes (e.g., cyanineprobes) can be selectively accumulated by the mito-chondria of living cells [75]. In 2011, our group devel-oped cyanine probes 33 for imaging 1O2 and 34 forimaging O3 in the mitochondria of living cells [76,77].Probe 33 comprised cyanine as fluorophore and histi-dine (His) as 1O2

�selective modulator. To constructprobe 34, we chose tricarbocyanine as NIR fluorescentdye and L-tryptophan (Trp) as an O3-indicator based ona twisted intramolecular charge transfer (TICT) mecha-nism.

In 2007, Nagano et al. selected 4-aminophenyl arylether and 4-hydroxylphenyl aryl ether as selectivemodulators based on rhodamine dye to synthesize fluo-rescent probes 35a and 35b for selective detection ofhROS in mitochondria of living cells [78]. 35a and 35breacted with intracellular hROS selectively over otherROS as due to the strong oxidation capacity of hROS.

3.3. Nuclear-targetable fluorescent probesThough various fluorescent probes targeting to themitochondria are extensively explored with an explicitmechanism, how to target other organelles (e.g., nuclearand lysosome) is still not definite and more studies areneeded to design targetable fluorescent probes for otherorganelles. There are a few reports on nuclear-targetablefluorescent probes and lysosome-targetable fluorescentprobes, which are only validated by confocal microscopyexperiments. More studies need to elaborate further the

mechanism of how the fluorescent probes accumulatewithin the corresponding organelles. As reported byUchiyama et al., through a nano fluorescent polymericthermometer 36 (Fig. 10), the distribution of intracel-lular temperatures in the nucleus and the centrosome ofa COS7 cell differed and showed a significantly highertemperature than in the cytoplasm [79]. As there aremany cellular events in the nucleus (e.g., DNA replica-tion, transcription and RNA processing) and its struc-tural separation by the nuclear membrane thatcontribute to the temperature difference [79], ISMRSmay also maintain different levels in the nucleus andother organelles.

In 2011, Chang et al. developed a nuclear-targetablefluorescent H2O2 probe 37, which revealed a link be-tween longevity-promoting sirtuin protein and enhancedregulation of nuclear ROS pools [80]. Probe 37selectively accumulated in the nuclei of a variety ofmammalian cell lines and showed selective response tosub-cellular H2O2 changes, which proved to be suitablefor monitoring sirtuin-mediated oxidative stress re-sponses. However, the molecular mechanism of 37localized in the nucleus and the effects of temperature onthe homeostasis of ISMRS remain unclear and deservefuture investigation.

3.4. Lysosome-targetable fluorescent probesThe proton concentration within mammalian cells canvary in the range 10–1000 nM [12]. Many biologicalevents and processes can be monitored by measuringlocal intracellular fluctuations in the value of pH. Themechanism of fluorescent probes accumulating in acidicvesicles is through incorporation of chemical tags thattend to be reactive toward protons in the protonatedform (e.g., anilines). They should be weak or non-fluorescent in the non-protonated form at pH 7.4 andbecome highly fluorescent in the protonated form underacidic conditions (pH < 6). However, limitations of thecurrently available pH probes include low sensitivity andexcitation profiles in the ultraviolet region that candamage living samples and cause interfering autofluo-rescence from native cellular molecules. Also, very fewacidic fluorescent probes are considered to be desirablefor studying acidic organelles, which is a bottleneck incell biological or medical studies.

Our group designed NIR fluorescent probe 38 forimaging and detecting Cu2+ (Fig. 11) [81]. Probe 38effectively avoided the fluorescence quenching for theparamagnetic nature of Cu2+ via its strong bindingcapability toward Cu2+ in living cells with good selec-tivity, high sensitivity, good photostability and the abilityto work within the acidic pH range. It was interesting tofind that synthesized fluorescent probe 38 tended toaccumulate in lysosomes.

In 2009, Kobayashi et al. reported a series of acidicpH-sensitive fluorescence probes 39 based on BODIPY



bearing hydrogen, methyl or ethyl substituents on theaniline nitrogen for selective molecular imaging of viablecancer cells [82]. They were constructed by targeting themonoclonal antibody into carboxylic groups on BODIPYwith the human epidermal growth factor receptor type 2(HER2). They could be internalized via the endosomal-lysosomal degradation pathway after binding to HER2.They were almost non-fluorescent in the extracellularenvironment (at pH 7.4) and became highly fluorescentin the protonated form under acidic conditions (pH < 6).

Ying et al. similarly synthesized cell-permeableBODIPY analogs 40 for selective labeling and monitor-ing pH changes of lysosomes in living cells [83].Fluorescent imaging showed that probe 40 was suitablefor lysosome labeling, and for non-invasive monitoringof lysosomal pH changes during physiological andpathological processes.

Gao et al. construed a series of nanoprobes 41 totarget the acidic endosomal/lysosomal compartments.They were prepared based on the supramolecularself-assembly of ionizable block copolymer micelles con-taining tetramethyl rhodamine (TMR) as pH-insensitivereference dye [84]. The NPs can be selectively activatedat various pH values, especially when in endocyticcompartments (e.g., early endosomes or lysosomes inhuman cells).

3.5. Cell-trappable fluorescent probesThere is emerging evidence that improvement of cellularretention of fluorescent probes in organelles or in cytosolis a practical, promising strategy to improve sensitivity offluorescent probes. A repertoire of cell-trappable ISMRS-sensitive fluorescent probes has therefore been developedand applied to cell imaging [85–94]. Typically, there aretwo approaches to increase cellular retention of fluo-rescent probes – ester functionalization [85–93] andpolymer conjugation [94] – which are achieved throughmodification of carboxylic moieties to acetoxymethyl(AM) esters or conjugation to polymer. On entering intothe cells, the ester groups are hydrolyzed by non-specificintracellular esterases. Cell-trappable ISMRS-sensitivefluorescent probes show superior intracellular retention,as negatively charged tetracarboxylates or polymercannot penetrate cellular membranes, so the fluorescentprobes are restricted to the cytosol and the nucleus,

Turn on Turn off

(a) (

Figure 13. Mechanism of reve


which, in turn, increase sensitivity and make thempossible to visualize much lower concentrations of spe-cific ISMRS. This advantage contributes to making itpossible to perform longer observation in living cells.

In 1981, Tsien developed the first cell-trappable fluo-rescent probe 42 (Fig. 12) for Ca2+ by means of esterfunctionalization, and it had unusual specificity for Ca2+

over Mg2+, H+, Na+ and K+ [85]. Since that time, manyefforts have been made to convert the probes into thecorresponding ester derivatives or to conjugate probes topolymers.

In 2010, Lippard et al. developed an esterified ligand forZn2+, which could be effectively trapped by cells [86].Probe 43 was poorly emissive in the off-state but exhibitdramatic increases (10-fold) in fluorescence upon Zn2+

binding over other biologically relevant metal ions. It wascell-membrane-permeable until cleavage of its estergroups by intracellular esterases produced a negatively-charged acid form that could not cross the cell membrane.

In 2011, Taki et al. developed a cholesterol-conju-gated fluorescence Zn2+ probe 44 capable of detectingextracellular Zn2+ in the vicinity of the plasma mem-brane [87]. In the absence of Zn2+, the fluorescence of44 is nearly quenched (U < 0.005) because of the PETprocess and is enhanced (�45-fold) upon coordinationwith Zn2+ (U = 0.18). This result indicated that thecholesterol moiety strongly interacted with membranelipids, resulting in the probes being located at theextracellular region because of the high polarity of thecarboxyl group.

Fluorescent probe 45a was reported by Hempel et al.[88]. It was generated by conjugating diacetate ester to2 0,7 0-dichlorodihydrofluorescein (DCFH). After it waspassively diffused into cells, the two acetate groups werecleaved by intracellular esterases to yield DCFH. To im-prove cellular retention of the oxidation product, achloromethyl derivative of 45a, 5-(and 6-)chloromethyl-2 0,7 0- dichlorodihydrofluorescein diacetate (CM-DCFHDA) 45b, was subsequently prepared by Jackson et al.[89]. The chloromethyl group of 45b allowed for cova-lent binding to intracellular thiol components, resultingin retention of CM-DCF within the cell for longer timeintervals. Accumulation of CM-DCF in cells can bemeasured on the basis of an increase in fluorescence at530 nm on excitation at 488 nm.

Turn onTurn off

b)

rsible fluorescent probes.

O

S S

H

HO OH

51

O

N

OHHO

CO2H

O.

+

52

Se

NN+

I-

53

OHS

N

S

O

54

N

N

O

O-

NN

NN R

R+

++

R= n-octyl

55Figure 14. Structures of reversible fluorescent probes 51–55 for detecting ISMRS.

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In 2010, Chang et al. developed two new probes, 46and 47, which took advantage of multiple maskedcarboxylates to increase cellular retention and hencesensitivity to low levels of H2O2 [90,91]. In their ester-protected forms, the 46 and 47 dyes are more lipophilicthan their carboxylate counterparts and can readilyenter cells. Once inside cells, the protecting groups arerapidly cleaved by intracellular esterases to produce theiranionic carboxylate forms, which are effectively trappedwithin cells because they cannot pass back through theplasma membrane. The increased retention of 46 and47 leads to their enhanced sensitivity to H2O2 throughirreversible boronate-oxidation events.

In particular, probe 46 was utilized to interrogate thecellular mechanisms involved in trafficking H2O2 duringgrowth-factor signaling. Certain classes of aquaporinwater channels, the aquaglyceroporins and unorthodoxaquaporins but not classic aquaporins, can enhance theuptake of extracellularly-produced H2O2 and regulateintracellular signal transduction. This work representedthe first study revealing that aquaporins can mediateboth H2O2 transport and signaling in mammalian cellsand had broad implications for H2O2 biology in processesranging from cell migration to wound repair [90].

In parallel work using 46 for imaging, the roles ofH2O2 in the self-renewal of neural stem cells were ex-plored. It was discovered that adult hippocampal pro-genitor cells (AHPs) required basal generation of H2O2

for their normal growth and proliferation in cell cultureand in vivo and determined that the NOX2 enzyme andphosphatase PTEN are molecular sources and targets ofH2O2, respectively. This study provided evidence thatH2O2 is a physiological regulator in living organisms,demonstrating that controlled ROS levels are needed tomaintain specific cell populations [91].

In 2009, Nagano et al. found that fluorescein-basedfluorescent probes 48 and 49 containing the iminodi-acetic acid group (IAG) showed superior intracellularretention, which could be utilized to increase the sensi-tivity of fluorescence probes [92]. Probe 48 was sensitiveto NO and 49 gave selective response to hROS. Theywere successfully applied to cell imaging.

In 2010, Lippard et al. developed cell-trappable NO-sensitive probes 50a and 50b by incorporating a estermoiety and a dextran moiety into fluorescein-based

Figure 15. Mechanism of multi-fu


symmetrical ligands [93,94]. Cu(II) complexes as NO-specific fluorescent probes have been illustrated in aprevious report. Both 50a and 50b responded rapidlyand selectively to NO over other ROS and RNS in mouseolfactory bulb slice. Considering the dynamic changes ofROS, RNS and pH, it is of paramount significance todesign multi-functional fluorescent probes that couldsimultaneously monitor several metabolites [95].

3.6. Challenges for sub-cellular targetable fluorescentprobesImaging the homeostasis of ISMRS by use of targetablefluorescent probes is critical for further research on cel-lular function. However, considering the complexity ofthe microenvironments in different organelles, severalfactors should be taken into account during design oftargetable fluorescent probes:

(i) the temperature in mitochondria and nuclei isobviously higher than other organelles, which has asignificant effect on fluorescence, so temperature-insensitive fluorescent probes are preferred;

(ii) the pH value in acidic organelles [e.g., lysosomesand endosomes (pH range 4.5–6.0)] is lower than inother organelles;

(iii) the relative level of ISMRS is different in variousorganelles (e.g., H2O2 and O2

�� are estimated to be in theratio of around 100:1 in the mitochondrial matrix butaround 1000:1 in the cytosol). The studies of such fac-tors are still not explicit. Only if they are studied deeplycould the complex mechanism on the role on ISMRS inorganelles be fully unraveled. The perfect targetablefluorescent probes should be single-factor responsive, notmulti-factor responsive, so as to assure a proper fluo-rescent signal. Future design will focus on the elaboratemicroenvironments of various organelles in living cellsand environmentally-sensitive chemical tags that can bespecifically targeted to sub-cellular microenvironments.

4. Reversible fluorescent probes for imaging ofredox events in living cells

An important development of functional fluorescentprobes in living cells would be the possibility to monitorthe dynamic redox homeostasis by reversible fluorescent

ISMRS indicator

ISMRS indicator

Reference dye

nctional fluorescent probes.

NN

O

OO

OO

N

O

O

NN

H2N

56

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OO

O

O

O

O

OH

N

O

O

O

O

BB

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OO

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probes. Redox homeostasis is critical for proper cellularfunction and metabolism. Changes to the intracellularredox environment would cause oxidative stress, whichhas been implicated in a variety of diseases, includingcancer, ischemia-reperfusion injury, and neurodegener-ation [96,97,100,109,119]. The redox status of the cellis primarily a consequence of the precise balance be-tween the levels of ROS and reducing equivalents (e.g.,organoselenium) and the thiol-disulfide interchangereaction [96,119]. A hallmark of oxidative stress is theabnormal ratio of GSH to its disulfide in cells [97,119].

Currently, four redox processes are used for designingreversible fluorescent probes:

(1). the electron-loss and electron-capture process{e.g., ferrocene [98,99]};

(2). dehydrogenation and hydrogenation reaction{e.g., fluorescein [100], tetrathiafulvalene[101,102], hydroquinone-benzoquinone switch-ing [103–107], 2,2,6,6-tetramethyl-1-piperidin-oxyl [108,109], and NADH analogue [110–112]};

(3). oxygenation-deoxidation reaction {e.g., oxida-tion of organoselenium [113,114] or tellurium[115,116]}; and,

(4). thiol-disulfide interchange reaction [117–119].Modulating the fluorescence of reversible fluorescent

probes is achieved by reversible binding with ISMRSalong with the four redox processes (Fig. 13). Amongthem, several interesting small-molecule fluorescentprobes 51–55 that can measure intracellular redoxenvironments and related ISMRS have been developed[100,109,114,119,120] (Fig. 14).

Based on the dehydrogenation and hydrogenationreaction, Chang et al. devised fluorescent probe 51 forimaging reversible redox cycles in living cells by intro-ducing disulfide into fluorescein [100]. Bottle et al. de-signed a novel profluorescent nitroxide 52 based onfluorescein as a sensitive probe for the cellular redoxenvironment [109]. Probe 52 could sensitively quantifychanges of intracellular redox status, especially in givinga selective response to in vitro O2

�� over H2O2 and �OH.Inspired by the principal modes of enzymatic ROS

scavenging by the ascorbate-GSH cycle and the gluta-thione peroxidase (GPX) cycle, our group prepared NIRreversible fluorescent probe 53 for ONOO� and imagingof redox cycles in living cells [114].

Murthy et al. chose coumarin as fluorophore andconjugated a thiolate to its extended aromatic p systemas a sensitive fluorescent probe 54 to measure the thiol-disulfide equilibrium of biological systems reversibly andratiometrically [119]. The fluorescence emission of 54and its disulfide are different after excitation at 372 nmand 488 nm, which allow for ratiometric imaging of thethiol-disulfide dynamics (dynamic GSH/GSSH ratios).Probe 54 was applied to monitor the reversible redoxstatus of whole-cell lysates.


You et al. and Chen et al. developed Fe2+-selectivefluorescent bioimaging probes 55 [120] by introducingthe imidazolium-based ionic liquid pendants into thesquaraine skeleton and controlling the aggregationbehavior of squaraines by adjusting the alkyl chain at-tached to the imidazolium unit. In the absence of Fe2+,probe 55 was fluorescent. However, fluorescence disap-peared upon the addition of both FeCl2 and H2O2 thatgenerated �OH due to attack the �OH to the four-membered ring of squaraine. Moreover, the cells couldlight up renewably in 20 min after a fluorescence‘‘turn-off’’ response. These imidazolium-anchored squa-raines in combination with the Fenton Reaction couldperform ‘‘naked-eye’’ detection of the Fe2+ over othermetal ions in living cells.

In considering the reactive nature of ROS, RNS andRCS, the fluorescent probes must provide fast, selectiveand sensitive response to them. To give further reversibleresponse to them, there are few fluorescent probes tomeet this requirement, so the challenges in reversiblefluorescent probes include:

(1). developing fluorescent probes with more detec-tion cycles and better biocompatibility;

(2). design of highly specific fluorescent probes foronly one species; and,

(3). multi-functional probes that could simulta-neously monitor several reversible cellular pro-cesses.

5. Multi-functional probes for simultaneousimaging of multiple components in living cells

Another important development of functional fluores-cent probes in living cells would be the possibility toreport on ISMRS concentrations simultaneously withdifferent fluorescent probes for multi-modal or multi-parameter imaging. As far as the complex cellularenvironment is concerned, different fluorescent probesfor multi-modal or multi-parameter imaging should beassembled into a molecular cargo [e.g., chemically-modified nanomaterials or fluorophores (e.g. coumarinand rhodamine dyes)], which are collectively termedmulti-functional probes (Fig. 15).

To build such multi-functional probes is a great needfor chemists and biologists. As ISMRS are involved in thedynamic changes, some of them, especially ROS andRNS, are very reactive species that have short lifetimes of�ns [1–4]. Intracellular environmentally-sensitiveparameters, including intracellular viscosity, proteaseactivity and temperature, are closely connected withtransportation and diffusion of reactive metabolites (e.g.,ROS and RNS) [79]. Changes in them at cellular levelhave a great effect on diseases and pathologies, so everyparticular metabolite of ISMRS and intracellularenvironmentally-sensitive parameters need to be


monitored directly near the sites of production and ac-tion (in situ).

Recently, Lin et al. chose phenylenediamine as thereaction site of NO, the boronate group as theH2O2-responsive site and 7-hydroxycoumarin and rho-damine dyes as fluorophores to synthesize multi-func-tional imaging probe 56 that could simultaneouslyreport intracellular H2O2, NO, and H2O2/NO with adistinct fluorescence signal pattern of blue�black�black,black�black�red, and black�red�red, respectively[121] (Fig. 16).

Nanostructures provide a broad platform to designmulti-functional fluorescent probes due to their promi-nent engineerability [122–131]. Nanomaterials withunique properties (e.g., polymers, carbon nanotubes,magnetic NPs and noble-metal NPs) have shown greatpromise for cancer therapy.

So far, several nanoprobes have been utilized formulti-functional detection and therapeutic application inbiomedical imaging. Liu et al. fabricated dual-functionalnanoprobe 57 based on hydrophilic block copolymers fordetecting changes in temperature and Zn2+ [122].Nanoprobe 57 was achieved through polymerization ofa thermo-responsive block by incorporating quinoline-based Zn2+-responsive fluorescent probe (ZQMA) into thepolymer. At low temperature, nanoprobe 57 couldselectively bind Zn2+ over other metal ions, resulting inthe prominent fluorescence enhancement. Fluorescentimages of 57 in HeLa cells promoted the investigation ofthe effects by thermo-induced micellization and detectionconditions of intracellular Zn2+ fluxes.

Our group also designed several multi-functionalplatform and fluorescent probes with simultaneous tar-geting, therapeutic and imaging functions for studyingthe inter-conversion of ROS and therapeutic applications[123–131]. Bifunctional Au-Fe2O3 NPs exhibitedsimultaneous targeting, therapeutic and imaging func-tions in cancer [127]. The bi-photosensitizer molecularbeacon could report the specific DNA/RNA with highselectivity and generate 1O2 effectively in breast-cancercells, but not in normal cells, which holds great promisefor cancer diagnosis and therapy [128].

In 2011, Chang et al. selected acetyl-capped G5PAMAM dendrimers functionalized with boronate-cagedPeroxyfluor-1 fluorophores for H2O2 detection and semi-naphthorhodafluor dyes for pH sensing as the platformto construct 58 for simultaneously detecting intracellu-lar pH and H2O2 [132]. Probe 58 could give a simulta-neous response to changes in H2O2 and pH by usingdifferent excitations resulting in different emissions,which allowed for selective discrimination between H2O2

and pH changes in live RAW 264.7 macrophage cells.The ultimate development of functional fluorescent

probes in living cells would be the possibility to designmulti-functional probes to image simultaneously ISMRSand related intracellular environmentally-sensitive

parameters. We note that, during the assembly of multi-functional probes, the differences in the structures of thefluorescent probes for cations, ROS, RNS, RCS and RSSshould be taken into account. These fluorescent probesusually consist of a selective signaling moiety andfluorophores.

For metal-responsive fluorescent probes, the majorityof selective signaling moieties are amine-, ether- or thi-oether-rich receptors (e.g., dipicolylamine is frequentlyused as receptor for Zn2+). Activation or deactivationfluorescence is controlled by inhibition of PET by coor-dination of metals, which is a commonly employed sig-naling mechanism for fluorescence enhancement.

For proton-responsive fluorescent probes, thesignaling moieties are often anilines. The fluorescentprobes should be weak or non-fluorescent in thenon-protonated form but become highly fluorescent inthe protonated form under acidic conditions.

Different from metals and protons, ROS, RNS, RCS andRSS are relatively more reactive. For selectively detectingthem, the signaling moiety is often elaborately designedto give a fast, highly selective response.

6. Summary and prospects

The visualization of various ISMRS implicated in diversecellular processes requires the use of spectroscopicallydistinguishable fluorescent imaging reporters. Func-tional fluorescent and luminescent probes, includingratio fluorescent and luminescent probes, organelle-targetable fluorescent probes, reversible fluorescentprobes, multi-functional probes and correspondingnanoprobes substantially improve the performance offluorescent imaging reporters, which have an advantageover conventional fluorescent probes.

Furthermore, RSS are the emerging new species thatwere recently detected in living cells [133–137]. ISMRShomeostasis and signaling are important in the ageingprocess and associated diseases, but their exact rolesremain insufficiently understood.

Compared with conventional fluorescent probes,functional probes have the advantages of multi-functionality, precision and enormous flexibility,allowing for the integration of multi-modal reportingfluorescent signals. Conclusively, imaging of intracellu-lar ISMRS utilizing functional small-molecule fluorescentprobes and nanoprobes are among the trends in devel-opment of imaging probes. This new avenue to study theinter-conversion of ISMRS holds the promise of eluci-dating the biological roles of ISMRS in signal transduc-tion and regulating cellular function.

AcknowledgementThis work was supported by the National KeyNatural Science Foundation of China (21035003), the



National Natural Science Foundation of China(2122700521105059), the Specialized Research Fundfor the Doctoral Program of Higher Education of China(20113704130001) and the Program for ChangjiangScholars and Innovative Research Team in University.

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