5790 Chem. Commun., 2013, 49, 5790--5792 This journal is c The Royal Society of Chemistry 2013

Cite this: Chem. Commun.,2013,49, 5790

A near-infrared reversible and ratiometric fluorescentprobe based on Se-BODIPY for the redox cyclemediated by hypobromous acid and hydrogen sulfidein living cells†

Bingshuai Wang, Peng Li, Fabiao Yu, Junsheng Chen, Zongjin Qu and Keli Han*

We have developed a near-infrared (NIR) reversible and ratiometric

fluorescence sensor based on Se-BODIPY for the redox cycle

between hypobromous acid oxidative stress and hydrogen sulfide

repair. Real-time imaging shows that the probe is able to monitor

intracellular HBrO/H2S redox cycle replacement.

Hypobromous acid (HBrO) is an integral factor in biological hostdefense systems, with very similar chemical and physical propertiesto those of hypochlorous acid.1 Endogenous HBrO is formed by thereaction of H2O2 with Br� catalyzed by eosinophil peroxidase (EPO).2

In vivo, HBrO is a powerful oxidizer with effective antibacterialactivity, however, excessive generation of HBrO will injure organismsand lead to inflammatory tissue damage,3 and can result in manydiseases, such as arthritis, cardiovascular diseases, cancers, andasthma.4 The correlation between serum EPO and clinical severityin asthmatic patients provides major evidence that EPO levels inserum increase by 300% in asthmatic patients than in healthyindividuals.4–6 In light of the potential role of HBrO in thesediseases, it is significant to explore the physiological functions ofHBrO in biological tissues.

Upon the occurrence of HBrO-oxidative damage in cells, theantioxidative defense mechanisms of living organisms promptlybegin to protect the essential components of cells.7 Although hydro-gen sulfide (H2S) has been recognized as the third gasotransmitter,8 italso acts as an important endogenous antioxidant, which is enzyma-tically generated in many organs (heart, lungs, kidneys, brain, nervoussystem, etc.).9 The endogenous H2S can play an anti-inflammatory roleand can help in the recovery of the pulmonary function in asthmapathogenesis.8–11 The H2S concentration in serum decreases by 60%in asthmatic patients compared with healthy subjects.9 It is suggestedthat endogenous H2S has the potential to serve as an inhibitor or arepairer of HBrO-induced oxidative stress in vivo. Therefore, it isimportant to investigate the redox process between HBrO and H2Sin cells.

Due to the specificity and cumulative signaling effects offluorescence technology, many fluorescent probes have becomeimportant tools for detecting various species in bio-systems to studyphysiological and pathological processes.12 The redox cycle betweenHBrO and H2S is a very complicated process that requires reversiblefluorescent probes for visual detection of the intracellular HBrO/H2Sredox cycle. There has been increasing research interest in thedevelopment of H2S fluorescent probes,13 however, only a fewfluorescent probes have been reported by our group for the redoxprocess between reactive oxygen species and H2S.14 Furthermore,only limited fluorescent probes have been reported for HBrO detec-tion.15 In this communication, we have developed a highly sensitivereversible fluorescent probe diMPhSe-BOD for the redox cyclebetween HBrO oxidative stress and H2S repair.

In order to achieve the visualization of dynamic changes in theequilibrium of HBrO/H2S redox coupling, a suitable chemical toolwas designed and synthesized for monitoring the intracellular redoxcycles. Our design concept for realizing our goal is based on theoxidation and reduction processes of selenides.16 We chose theBODIPY dye as the fluorophore because of its good photostability,strong extinction coefficients, and high fluorescence quantum yield.Next we integrated the modulator (4-methyoxylphenylselenide,MPhSe) into the fluorescent platform through a styrene bridge. Thisapproach can extend the p-conjugation system of the fluorophoreand tune the fluorescent emission red-shift efficaciously.17 Moreover,the strong electron-donating properties of the Se group furtherfacilitate the fluorescence of the probe to the near-infrared (NIR)region. Compared with UV-visible light probes, NIR probes areoptimal for biological imaging because of their minimum photo-damage, autofluorescence of biological objects.13d,18 Previously, wehave developed a selenium-containing fluorescent probe Cy-PSe formonitoring cellular redox changes induced by peroxynitrite andglutathione.19 The present probe, diMPhSe-BOD, only responds toHBrO and then efficiently reacts with H2S in its reduced form. After‘‘selenide’’ is oxidized to ‘‘selenoxide’’ by HBrO, the fluorescence ofthe probe will blue-shift because of the electron-withdrawing effect of‘‘selenoxide’’. Once ‘‘selenoxide’’ is reduced to ‘‘selenide’’ by H2S, theprobe recovers its original form and the fluorescence red shifts again(Scheme 1). The changes in fluorescence wavelength result in the

State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical

Physics (DICP), Chinese Academy of Sciences (CAS), 457 Zhongshan Road, Dalian

116023, P. R. China. E-mail: klhan@dicp.ac.cn

† Electronic supplementary information (ESI) available: Experimental details andsupplementary data. See DOI: 10.1039/c3cc42313a

Received 30th March 2013,Accepted 30th April 2013

DOI: 10.1039/c3cc42313a

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This journal is c The Royal Society of Chemistry 2013 Chem. Commun., 2013, 49, 5790--5792 5791

probe behaving as a ratiometric fluorescent probe, which cancircumvent the effects of various factors, including polarity, probemolecule concentration, and its stability under photo-illumination.20

Given these advantages, the probe is expected to acquire positiveresults for the HBrO/H2S redox cycle.

The spectral properties of the probe were investigated undersimulated physiological conditions. diMPhSe-BOD exhibits themaximum absorption at 672 nm (e = 22 770 M�1 cm�1) and a short-wavelength shoulder at 618 nm (e = 16 300 M�1 cm�1) (Fig. S1a, ESI†).Based on the normalized absorption spectra (Fig. S1b, ESI†), therelative intensity of the short-wavelength shoulder increased, and themaximum absorption and the short-wavelength shoulder blue-shiftedto 655 nm (e = 16 700 M�1 cm�1) and 603 nm (e = 13 800 M�1 cm�1),respectively (Fig. S1a, ESI†). This process was accompanied by anobvious color change in the probe solution from green to blue(Fig. S1b, ESI† inset images). We selected the maximum emissionwavelength (711 nm) of diMPhSe-BOD as the testing wavelength.

diMPhSe-BOD shows weak fluorescence due to the heavyatom effect.20 Both the absorption and emission spectra ofdiMPhSe-BOD lie in the NIR region. After the reaction of10 mM diMPhSe-BOD and 100 mM HBrO, the fluorescencemaximum blue-shifted to 635 nm because the weaker electron-donating ability of ‘‘selenoxide’’ than ‘‘selenide’’ shortened thedonor–acceptor p-conjugated system (Scheme 1). The fluores-cence intensity at 635 nm was remarkably increased by 230-foldbecause the heavy atom effect of Se was weakened by the bindingof an oxygen atom;21 the fluorescence quantum yield increasedfrom 0.00083 to 0.206. The detection limit of our probe wasdetermined to be 0.97 mM under the testing conditions. AfterHBrO oxidation, the intensity of the emission spectra (675–750 nm) of the probe would decrease (Fig. 1a), however, anenhancement of the intensity of the fluorescence spectra in therange of 675 nm to 725 nm was observed as the excitationwavelength was 610 nm. Hence, we selected 670 nm (see Section4 in ESI†) as the excitation wavelength, because the emission ofdiMPhSeO-BOD was negligible at this excitation wavelength(Fig. S3, ESI†). The fluorescence response of diMPhSe-BOD toHBrO was collected in the fluorescence window from 685 nm to850 nm. The emission intensities of diMPhSe-BOD decreasedslowly with the addition of HBrO (Fig. S3, ESI†). The emissionintensity ratio (F635nm/F711nm) increased by 118-fold as the HBrOconcentration increased from 0 mM to 50 mM (Fig. 1a, inset).A linear relationship (R = 0.988) was obtained between the ratio

and HBrO concentrations in the given range. Therefore, the probediMPhSe-BOD can be applied in ratiometric detection of HBrO.

For assessing the specific nature of diMPhSe-BOD towardsHBrO, we investigated the effects of relevant physiological oxidants,including cumene hydroperoxide (CuOOH), tert-butylhydroperoxide(t-BuOOH), Fe3+, O2

��, H2O2, �OH, NO, ONOO�, 1O2 and HClO. Thefluorescence intensity ratios of diMPhSe-BOD that reacted withvarious oxidants for 60 min are shown in Fig. 1b and Fig. S4 (ESI†).After adding HBrO into the testing solution, the fluorescence ratio ofour probe increased and the fluorescence ratio enhanced from0.10 to 11.88 within 3 min. Obviously, the fluorescent noise wasmuch lower than the fluorescence signal induced by HBrO. Theseselectivity profiles showed that diMPhSe-BOD had good selectivityand sensitivity to HBrO under simulated physiological conditions.

The selectivity of diMPhSeO-BOD to the reductants wastested within 30 min. As shown in Fig. S6 (ESI†), diMPhSeO-BOD was quickly reduced (within 10 min) by H2S, and thefluorescence intensity ratio recovered to the original level. By con-trast, the ratios decreased slowly upon the addition of the otherreductants. The fluorescent ratios of diMPhSeO-BOD after reactingwith various reductants for 30 min are presented in Fig. 2a. H2Sreduced the fluorescent ratio from 13.2 to 0.66. However, themaximum ratio decrease induced by Na2S2O4 among other reduc-tants was only 22% of the blank value. Fig. S8 (ESI†) shows the

Scheme 1 Structure of diMPhSe-BOD and the detection mechanism ofdiMPhSe-BOD for the HBrO/H2S induced redox cycle. Inset image: left: fluores-cence image of 10 mM probe; right: fluorescence image of 10 mM probe + 100 mMHBrO in 20 mM pH 7.4 PBS (20% acetonitrile).

Fig. 1 Fluorescence spectra of diMPhSe-BOD (10 mM) with HBrO in differentconcentrations of 20 mM pH 7.4 PBS (acetonitrile 20%) (lex: 610 nm, HBrOconcentrations: 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, and100 mM). Inset: the relationship between the fluorescence intensity ratios (F635nm/F711nm) and the HBrO concentrations. F635nm and F711nm: fluorescence intensity of theprobe solution at 635 and 711 nm, respectively. (b) Fluorescence intensity ratio ofdiMPhSe-BOD that reacts with ROS for 60 min. F635nm and F711nm: fluorescenceintensity of the probe solution at 635 and 711 nm, respectively (lex: 610 nm).

Fig. 2 (a) Comparison of the fluorescence intensity ratio of diMPhSeO-BOD (10 mM)with the addition of different reductants after 30 min in 20 mM PBS (acetonitrile 20%)at pH 7.4 (lex: 610 nm). (b) Fluorescence ratio responses of diMPhSe-BOD (10 mM) toredox cycles. diMPhSe-BOD was oxidized by HBrO. After 10 min, the solution wastreated with H2S in an appropriate concentration. When fluorescence intensity at635 nm returned to starting levels, another portion of HBrO was added. The redoxcycles were repeated five times. All spectra were acquired in 20 mM PBS pH 7.4, lex =610 nm. The representation of the letters in Fig. 2: a, 50 mM HBrO; b, 45 mM H2S; c,100 mM HBrO; d, 45 mM H2S; e, 100 mM HBrO; f, 60 mM H2S; g, 100 mM HBrO; h, 60 mMH2S; i, 100 mM HBrO; and j, 60 mM H2S.

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5792 Chem. Commun., 2013, 49, 5790--5792 This journal is c The Royal Society of Chemistry 2013

fluorescence profile of diMPhSeO-BOD at different H2S concentra-tions. Both the fluorescence intensities at 635 and 711 nm returnedto their original levels. It is indicated that diMPhSeO-BOD can bereduced to diMPhSe-BOD by H2S. The fluorescence lifetimedecay of the reduction product overlapped completely with thatof diMPhSe-BOD (Fig. S7, ESI†). This result indicates that thefluorescence signal returned to the original state. The reducedprobe still possessed the same reactivity with HBrO as before.The redox cycles could be repeated at least five times (Fig. 2b)with only an 18% decrement in the ratio (F635nm/F711nm). Theseresults suggest that our probe diMPhSe-BOD can be used toselectively monitor the redox cycles between HBrO and H2Scontinuously and quantitatively. The detection limits for HBrOand H2S were determined to be 50 nM and 0.1 mM.

Based on the positive results, we next studied the bio-imagingapplications of diMPhSe-BOD for detecting the HBrO/H2S redoxcycle in a biological system. We chose the mouse macrophage cellline RAW264.7 as a bioassay model because the macrophage cellsactivated the generation of HOBr after exposure to EPO, hydrogenperoxide (H2O2), and bromide ions (Br�).22 Macrophage cells wereincubated with 5 mM probe for 10 min, cell images were takenthrough two collection channels ranging from 640 nm to 670 nmand 680 nm to 770 nm. The fluorescence intensity in the shorterchannel (FS) was weaker than that in the longer channel (FL) (Fig. S9aand b, ESI†). As mentioned previously, ratiometric detection canefficiently eliminate the interference of environmental factors in thecomplicated biological systems. The ratiometric cell image wasobtained using Olympus Micro FV10-ASW software with an averageratio of FS/FL = 0.6 to 0.8 (Fig. 3a). Upon the addition of H2O2

(20 mM), EPO (105 U mL�1), and KBr (50 mM) to the probe-loadedcells for 30 min, FS increased and FL decreased. Hence, FS becamemuch stronger than FL (Fig. S10a and b, ESI†) and the average ratioof FS/FL enhanced to 3.5–4.5 (Fig. 3b), which is in accord with theratio changes in solution. When the cells were incubated with 50 mMH2S for 20 min, the average ratio decreased to approximatelythe starting level (Fig. 3c), indicating that the oxidation productdiMPhSeO-BOD was reduced to diMPhSe-BOD. After another dose of

H2O2, EPO and KBr was added to the cells, the average ratioincreased again (Fig. 3d). All results demonstrate that our probediMPhSe-BOD can be applied in the ratiometric visualization of theHBrO/H2S redox cycle continuously in living cells. Co-staining withthe nuclear counterstain Hoechst (Fig. 3e and f) revealed that thesubcellular location of the probe was in the cytoplasm of the livingRAW264.7 cells. Bright-field transmission images confirmed that thecells are still viable (see ESI†).

In conclusion, we have successfully developed an NIR rever-sible and ratiometric fluorescent probe diMPhSe-BOD for theHBrO/H2S redox cycle based on the oxidation and reductionprocesses of selenides in solution and in living cells. Our probediMPhSe-BOD is highly sensitive and specific for the HBrO/H2Sredox cycle detection. Real-time imaging of the HBrO/H2S redoxcycle was successfully conducted with our probe diMPhSe-BODin macrophage cells. The experimental results show that theprobe has good permeability in living cells, and can monitorintracellular HBrO/H2S redox cycle replacement continuously.

This work was supported by NSFC No. 21273234, 21203192,and 2013CB834604.

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Fig. 3 Confocal fluorescence images of the redox cycles between HBrO and H2S inRAW264.7 cells. Macrophage cells were incubated with diMPhSe-BOD (5mM) for 10 minand then treated with various stimulants at 37 1C. Cell images were obtained at 635 nmexcitation wavelength and 640 nm to 670 nm and 680 nm to 770 nm emission bands.(a) Control; (b) probe-loaded cells incubated with H2O2 (20 mM), EPO (105 U mL�1), andKBr (50 mM) for 30 min; (c) cells in (b) incubated with H2S (50 mM) for 20 min; (d) (c) wastreated with a second dose of H2O2 (20 mM), EPO (105 U mL�1) and KBr (50 mM) for30 min; (e) overlay of images showing fluorescence from diMPhSe-BOD and Hoechstdye; (f) Overlay of bright-field, diMPhSe-BOD, and Hoechst dye images.

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