DOI: 10.1002/cplu.201402217

A Fluorescence Switch for the Detection of Nitric Oxideand Histidine and Its Application in Live Cell ImagingGandhi Sivaraman, Thangaraj Anand, and Duraisamy Chellappa*[a]

Introduction

Oxidative post-translational modification of proteins is cata-lysed by reactive oxygen species (ROS) and reactive nitrogenspecies (RNS).[1] Reactive oxygen and nitrogen species such asnitric oxide (NOC), superoxide (O2�

C), hydroxyl (HO�C) and peroxy(ROO�C) radicals, hydrogen peroxide (H2O2), singlet oxygen (1O2)and peroxy nitrite (ONOO�) are signalling molecules requiredfor normal cellular function. Among these species, nitric oxideis one of the signalling molecules in human beings.[2] Nitricoxide (NO) is a diatomic free radical that is extremely short-lived in living systems. It has been recognised as an intra- andextracellular messenger molecule in the immune system.[3, 4]

Furthermore, nitric oxide plays a vital role in human physiologysuch as vascular homeostasis,[5] neurotransmission,[6] host de-fence mechanism,[7] inhibition of platelets,[8] smooth-musclecell propagation[9] and attachment of leukocytes to the endo-thelium.[10] Lower-level production of NO in the body leads tohypertension, arteriolosclerosis and impotence, whereas over-production of NO is connected with diabetes,[11] stroke,[12]

septic shock,[13] multiple sclerosis[14] and cancer.[15] In view ofthese facts, several direct and indirect methods such as chemi-luminescence, electrochemical sensors[16] and electron para-magnetic resonance[17] techniques have been designed toquantify the NO levels. Classical techniques include electronparamagnetic resonance (EPR) spectroscopy. Electrochemicaltechniques lack the sensitivity and spatial resolution for thedirect detection of NO in live cells. Fluorescence is one of themost useful analytical tools in analysis of biological species be-cause fluorescence imaging of analytes offers an attractiveplatform for monitoring biological processes in high resolu-

tion.[18–21] Organic-molecule-based fluorescence sensors fornitric oxide have not found widespread application in biologi-cal systems as their fluorescence relies on their NO oxidised re-action products.[22–24] To overcome these issues, transition-metal-mediated NO sensors have been developed in recentyears.[25, 26] Nevertheless, metal-complex-based sensors havelimitations such as water insolubility and low sensitivity thatprohibits their biological applications. Complexes incorporatingfluorophore ligands for binding the metal ions such as iron,cobalt and ruthenium can react with NO to display fluores-cence enhancement. This has several disadvantages like re-duced turn-on fluorescence emission with NO and air sensitivi-ty; it is not preferred for real-time analysis of NO in biologicalcells. A strategy in which nonfluorescent paramagnetic CuII

complexes bearing fluorescent ligands are converted into dia-magnetic CuI by the reaction of NO with CuII to restore fluores-cence has been adopted to reduce these disadvantages.[27–32]

The fluorescent detection of amino acids[33–34] is currently of in-terest because of its wide applicability in biological samples in-cluding live cells.

Among the amino acids, histidine (his) is one of the essentialamino acids; it plays an important role as a neurotransmitterand a reduced level of histidine leads to long-lasting kidneydisease. An amplified level of histidine can result in asthmaand progressive liver cirrhosis.[35] The development of metal-ion-based turn-on fluorescent sensors for histidine has beenwidely reported.[36–39] Recently, rhodamine-dye-based sensingsystems were investigated extensively because of their largemolar extinction coefficient and visible excitation and emissionmaxima. The high fluorescence quantum yield of rhodaminedyes facilitates their utility as effective chromogenic and fluo-rogenic sensors for metal ions and small molecules.[40–43] Re-cently, rhodamine-based turn-on fluorescent NO sensors weredeveloped by utilizing spirolactam as a trigger.[44] By utilisingthe advantages of rhodamine dyes and a copper-ion-reductionstrategy with NO,[45] a new sensing system comprising rhoda-

A rhodamine-based copper complex as a selective, sensitiveturn-on fluorescent chemosensor for NO/histidine has been de-veloped. A conspicuous fluorescence enhancement was ob-served in the presence of nitric oxide/histidine. The probe wasspecific towards NO over other reactive oxygen species and re-active nitrogen species. The probe showed selective fluores-cence enhancement with histidine; the other naturally occur-

ring amino acids did not result in fluorescence enhancement.EPR and ESIMS studies clearly showed that NO-induced reduc-tion of copper ions leads to the fluorescence enhancement.The viability of the probe for fluorescent imaging of nitricoxide and histidine in living cells has been demonstrated bymeans of confocal laser scanning microscopy experiments.

[a] Dr. G. Sivaraman, T. Anand, Prof. Dr. D. ChellappaSchool of ChemistryMadurai Kamaraj UniversityMadurai, Tamil Nadu 625218 (India)E-mail : dcmku123@gmail.com

Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cplu.201402217.

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mine dye and CuII ions was developed. Wu and Yang et al.[46]

reported that a sensor molecule comprising rhodamine B dyeappended to 2-hydroxynaphthaldehyde by means of an iminelinkage shows an observable colour and fluorescence changeonly for HgII and exhibits only a colorimetric response for CuII

ions. The nonfluorescent nature of the CuII–rhodamine com-plex can be utilised for the turn-on detection of NO. It is clearfrom the data that rhodamine 6G dye derivatives are muchmore sensitive than rhodamine B derivatives as fluorescent re-sponsive probes. Recently, Gao and co-workers[47] developeda new HgII-detecting system containing 2-hydroxy-1-naphthal-dehyde and rhodamine 6G thiohydrazide that also showeda colorimetric change for CuII. Herein, we report the synthesisof rhodamine 6G bearing 2-hydroxy-1-naphthaldehyde (RDN)and their CuII (RDN–CuII) complexes, which displayed a fast andselective response to NO over other biologically active oxygenand nitrogen species.

Results and Discussion

Probe RDN was synthesised by the single-step condensation ofrhodamine 6G hydrazide with 2-hydroxy-1-naphthaldehyde inthe presence of acetic acid (Scheme 1). The structure of RDNwas confirmed by analytical techniques (Figure S1–S3 in theSupporting Information).

RDN in spirolactam form remains colourless and nonfluores-cent. The Job plot analysis from UV/Vis absorption titration ex-periments confirmed the 1:1 complexation of RDN with Cu2 +

(Figure S4). The stoichiometric binding of CuII with RDN wasfurther established by ESIMS spectra and the peak at m/z 663.96 corresponded to [RDN+Cu2++H2O�H]+ . RDN did not showany observable fluorescence when excited at 480 nm. After theaddition of Hg2+ ions the fluorescence intensity at 548 nm

(Ff = 0.88) increased immensely. However, CuII did not showany fluorescence even though RDN displayed an absorbancearound 527 nm with CuII, which is characteristic of the ring-opened form of rhodamine 6G dyes. This nonfluorescentnature of the RDN–CuII (Ff = 0.0047) species is attributable tofluorescence quenching as a result of the paramagnetic natureof copper ions.

The association constant of RDN with Cu2 + was found to be4.09 � 102

m�1 on the basis of the UV/Vis absorbance titration

experiments. This gives an opportunity to examine the restora-tion of fluorescence by reducing CuII with NO. The ability ofRDN–CuII to detect nitric oxide was investigated by UV/Vis ab-sorption spectra and emission spectra. We treated RDN–CuII in10 mm phosphate-buffered solution (pH 7.54) with an excessamount of NO. No change in the absorbance shift was record-ed whereas the fluorescence enhancement emerged immedi-ately at 548 nm (Ff = 0.792) when excited at 480 nm. The fluo-rescence intensity at lmax = 548 nm increased steadily duringaddition of NO to RDN–CuII within 15 minutes (Figure 1). When

the fluorescence response of the RDN–CuII probe for ROS andRNS species including NO, H2O2, ClO� , NO3

� , NO2� , ROO� ,

ONOO, O2, tBuOOH and HO� under physiological conditionswas investigated, only NO induced an increase of fluorescencewhereas all the other species did not affect the intensity offluorescence of the RDN–CuII probe (Figure 2). Hence, theRDN–CuII probe is selective for NO. The quenched fluorescenceintensity of the spiro-ring-opened rhodamine fluorophore isexpected to be restored on reduction of the copper(II) centreby nitric oxide.

The X-band EPR studies confirmed the reduction of the CuII

centres in the RDN–CuII complex by nitric oxide. RDN–CuII dis-played characteristic Cu2 + EPR spectra in ethanol. At roomtemperature, RDN–Cu showed EPR bands at 2.22, 2.02, whichvanished upon addition of NO (Figure S11). This EPR responseof NO clearly shows the reduction of the paramagnetic CuII tothe diamagnetic CuI centre with concomitant conversion ofNO to NO+ . In the ESIMS spectrum of the NO-treated solution

Scheme 1. Synthesis of RDN and RDN–CuII.

Figure 1. Time-dependent fluorescence enhancement of RDN–Cu (50 mm) asa result of the addition of NO (1 mm ; 20 equiv) in 100 mm phosphate buffersolution (pH 7.54) (lex = 480 nm).

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of RDN–CuII, the observed intense peak at 610.20 clearly showsthe formation of [RDN+NO�2 H]� and reduced CuI species.(Figure S7). The lower detection limit of NO was found to be inthe nanomolar range. The new peak at 1380 cm�1 in the IRspectrum of NO-treated RDN–CuII infers that NO appends tothe ring-opened probe RDN. The change in copper coordina-tion after the addition of NO can be easily explained by com-paring the IR spectra of RDN–CuII with that of RDN–CuII+NO.The observed shift in carbonyl stretching frequencies of RDN–CuII and RDN–CuII+NO implies the liberation of free RDN bythe dissociation of RDN–CuII. The hydroxyl stretching frequencyof RDN–CuII+N also supports the detachment of RDN–CuII andsubsequent release of ring-opened RDN (Figures S13 and S14).

Copper complexes are widely used for the differentiation ofhistidine from other amino acids.[48–50] This prompted us to ex-amine whether the RDN–CuII ensemble can be utilised as a pro-pitious turn-on fluorescent sensor for histidine (His). The fluo-rescence of RDN–CuII increased significantly upon addition ofhistidine. Fluorescence titration profiles of RDN–CuII (50 mm)with different concentrations of His (0–50.0 mm) indicate thatthe fluorescence intensity increases (Ff = 0.74) gradually withincreasing concentration of His (Figure 3). The detection limitof His was found to be 30 nm from the fluorimetric titration ofRDN–CuII with histidine. To examine the selectivity of RDN–CuII

in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)(20 mm, pH 7.4, containing 0.5 % dimethyl sulfoxide (DMSO) ascosolvent) for histidine, the remaining 19 naturally occurringamino acids such as tryptophan (Trp), asparagine (Asn), lysine(Lys), leucine (Leu), isoleucine (Ile), methionine (Met), threonine(Thr), tyrosine (Tyr), valine (Val), aspartic acid (Asp), alanine(Ala), serine (Ser), glutamine (Gln), glutamic acid (Glu), glycine(Gly), phenylalanine (Phe), cysteine (Cys), proline (Pro) and argi-nine (Arg), (10 equiv.) were added and the fluorescence intensi-ty was measured. The addition of these amino acids did notresult in any apparent fluorescence enhancement includingthat of Cys (Figure 4).

The interaction between His and RDN–CuII was further stud-ied by ESIMS spectroscopy. After addition of His, the peak atm/z�663.96 completely vanished and a peak at m/z�581.50was observed, which was attributed to the [RDN+H]+ probe.This observation clearly points out that the formation of a histi-dine complex with Cu2+ led to ring-opened of RDN. Thestrong binding capability of the imidazole group of His to thecopper ion caused the high selectivity of the complex for Hisover other amino acids. The binding constant of RDN with CuII

is much lower than that of the formation constant of His withCu2+ .[51] Hence the formation of the [Cu(His)2] complex favoursthe formation of the RDN–CuII complex (Scheme 2). The addi-tion of histidine to RDN–CuII leads to a larger enhancement offluorescence. This is owing to binding of the histidine amineand imidazole groups.

Figure 2. Fluorescence emission spectra of RDN–Cu (50 mm) in 100 mm

phosphate buffer (pH 7.54) in the presence of various RNS and ROS species(1 mm) after 15 minutes of addition.

Figure 3. Concentration-dependent fluorescence enhancement of RDN–Cu(50 mm) on the addition of various amounts of histidine in 100 mm phos-phate buffer solution (pH 7.54) (lex = 480 nm).

Figure 4. Fluorescence emission spectra of RDN–Cu (50 mm) in 100 mm

phosphate buffer (pH 7.54) in the presence of various amino acids(5 � 10�6

m) after five minutes of addition.

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It is further supported by the observation that addition ofhistamine to RDN–CuII also shows a fluorescence enhancementlike that of histidine, which indicates that the binding of theimidazole group with CuII leads to fluorescence enhancement.This is also evidenced from the treatment of Na,Nim-tert-buty-loxycarbonyl (Boc)-protected histidine, which did not result inany enhancement of fluorescence. The addition of proteins likebovine serum albumin (BSA) and human serum albumin (HSA)did not lead to any fluorescence enhancement (Figure S10).The formation of the [Cu(His)2] complex was further examinedby the IR spectral studies on RDN–CuII- and RDN–CuII-treatedhistidine (Figure S15). The IR spectrum of RDN–CuII completelychanged after addition of histidine; a dramatic change in thehydroxyl-group and carbonyl-group stretching frequencieswere observed. The comparison of the IR spectra of histidine-treated RDN–CuII with pure RDN reveals that RDN is present inthe ring-opened form (see the Supporting Information) ratherthan in the original spiro-ring closed form.

To assess the ability of RDN–CuII to operate in biosystems, itsresponse towards NO in live cells was studied. The candida al-bicans cells, when incubated with 5 mm RDN–CuII in phos-phate-buffered saline (PBS) for 30 minutes at 37 8C, did not ex-hibit any intracellular fluorescence. The probe in an extracellu-lar medium was washed with PBS three times. Then, theprobe-treated cells were incubated with NO for 15 minutes at37 8C and then the fluorescence signal in the cells was moni-tored; the red fluorescence appeared in the cells (Figure S12).Bright-field measurements confirmed that the cells were viablethroughout the imaging experiments. These experiments sug-gested that the RDN–CuII probe can be used for detection ofNO within living cells. Recently, Meng et al.[52] reported twosimilar rhodamine-based fluorescence probes for the turn-ondetection of nitric oxide that was assisted by CuII ions in thenanomolar range and was further applied to living cell imag-ing. Similar to their report, our case also showed the nitrosa-tion of the rhodamine probe.

The applicability of the RDN–CuII probe for imaging endoge-nously produced NO in RAW 264.7 murine macrophages wasstudied. Inducible nitric oxide synthase (iNOS) was induced bythe treatment of 1.25 mg mL�1 of lipopolysaccharide (LPS) and1000 U mL�1 of interferon-gamma (IFN-g) in raw cells for twohours. The cells were then incubated with 5 mm RDN–CuII. Afterthe incubation of the probe the cells were washed again withPBS buffer and analysed by fluorescence microscopy. Red fluo-rescence was observed from the macrophages whereas thecells without the treatment of LPS and IFN-g did not yield anyobservable fluorescence. It is clearly shown that RDN–CuII is ap-propriate to image endogenously produced nitric oxide(Figure 5). The time-dependent fluorescence response of RDN–

CuII with nitric oxide and histidine were measured. With nitricoxide, the fluorescence intensity of RDN–CuII enhanced withinfive minutes, whereas with histidine the intensity enhancedonly after 20 minutes. This gives the opportunity to distinguishthe presence of both. The RDN–CuII probe is not only sensitiveto nitric oxide but also histidine. The intracellular imaging ofNO and histidine separately in the cells does not pose a prob-lem. The main potential disadvantage of the probe is its inabil-ity to differentiate histidine and NO inside the living cellsduring imaging as both turn on the fluorescence with theprobe. If both NO and histidine are present together, the imag-ing of NO is preferred over histidine because NO has a fasterresponse towards RDN–CuII than histidine. In other words, NOundergoes a fast reaction with the CuII centre of the probe toreduce it to CuI. Being d10, CuI has no crystal-field stabilizationenergy and gets detached from the probe to restore the fluo-

Scheme 2. Schematic representation of the sensing behaviour of RDN–CuII

for NO and histidine.

Figure 5. Fluorescent imaging of endogenous NO without and with stimula-tion by lipopolysaccharide (LPS; 1.25 mg mL�1) and interferon-gamma (IFN-g ;1000 U mL�1) in RAW 264.7 macrophages by the RDN–CuII probe: A) bright-field images of RAW 264.7 macrophages without treatment of LPS and IFN-g ; B) fluorescence images of RAW 264.7 macrophages without treatment ofLPS and IFN-g ; C) bright-field images of RAW 264.7 macrophages after treat-ment of LPS and IFN-g for 24 h; D) fluorescent images of RAW 264.7 macro-phages after treatment of LPS and IFN-g for 2 h.

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rescence. Live cell imaging with MCF-7 cells for histidine usingRDN–CuII was carried out. It should be noted that cells treatedwith the probe alone result in only very weak fluorescence en-hancement. But strong fluorescence enhancement is observedwhen the probe-treated cells are incubated with histidine forten minutes. This clearly shows that the probe is viable for thedetection of histidine (Figure 6)

To get the information about the geometry, electronic struc-ture and photophysical properties of RDN, RDN–CuII, the ring-opened form of RDN, and NO-substituted RDN, DFT-B3LYP/6-311G- or LANL2DZ-level calculations were carried out using theGaussian 09 program.[53] The geometrical parameters of thecompounds coincide well with the literature values. In RDN–CuII, the bond lengths of Cu�Oare 2.016 (amide carbonyl) and1.905 � (phenolic oxygen) andthe bond length of Cu�N is2.117 �.

These bond lengths coincidewell with reported values.[54] Fur-thermore, time-dependent (TD)DFT studies on RDN, RDN–CuII,ring-opened RDN, and NO-sub-stituted RDN support the ob-served photophysical change.The frontier molecular orbitalsclearly indicate that the molecu-lar orbitals of ring-opened RDNand NO-substituted RDN differfrom probe RDN (Figures 7 and8). The DFT-optimised geometryof the RDN–CuII complex has dis-torted-tetragonal geometry. Thisfurther gives evidence for theobserved EPR spectrum (see theSupporting Information). The op-timised structures are shown inFigure S19.

Conclusion

In summary, a rhodamine-bear-ing 2-hydroxy-1-naphthaldehydeprobe (RDN) has been synthes-ised. The RDN–CuII complex wasspecific towards NO/histidine. Itprovides turn-on fluorescenceand high selectivity with NO andhistidine over other amino acidsand ROS and RNS species. Fluo-rescence imaging shows thatRDN–CuII could be used for thedetection of NO and histidine inliving cells.

Figure 6. Fluorescent imaging of MCF-7 cells : A) fluorescence image of con-trol cells after incubation with RDN–Cu (10 mm) ; B) bright-field transmissionimage of cells incubated with RDN–Cu (10 mm) ; C) fluorescence image ofcells supplemented with RDN–Cu and histidine (100 mm) for 10 min; D) fluo-rescence imaging of cells without the supplement of histidine. Excitationperformed at 480 nm.

Figure 7. Frontier molecular orbitals of RDN and ring-opened RDN.

Figure 8. Frontier molecular orbitals of RDN–CuII and NO-substituted RDN.

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Acknowledgements

G.S. thanks UGC for a research fellowship. G.S. , T.A. and D.C. ac-knowledge DST-IRHPA, FIST and PURSE for funding and instru-mental facilities. We thank Prof. S. Krishnaswamy, School of Bio-technology, for help.

Keywords: amino acids · cell imaging · fluorescence · nitricoxide · rhodamine

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Received: July 10, 2014

Revised: September 3, 2014

Published online on && &&, 0000

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G. Sivaraman, T. Anand, D. Chellappa*

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A Fluorescence Switch for theDetection of Nitric Oxide andHistidine and Its Application in LiveCell Imaging

Let there be light : A rhodamine-basedcopper complex as a selective, sensitiveturn-on fluorescent chemosensor fornitric oxide/histidine has been devel-oped (see scheme). The probe displayeda fast and selective response to NO over

other biologically active oxygen and ni-trogen species. The mechanism wasstudied and the viability of the probefor fluorescent imaging of nitric oxideand histidine in living cells was demon-strated.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemPlusChem 0000, 00, 1 – 7 &7&

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