This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 11507–11509 11507

Cite this: Chem. Commun., 2011, 47, 11507–11509

A copper(II) rhodamine complex with a tripodal ligand as a highly

selective fluorescence imaging agent for nitric oxidew

Xiaoyue Hu, Jian Wang, Xiang Zhu, Dapeng Dong, Xiaolin Zhang, Shuo Wu and

Chunying Duan*

Received 6th July 2011, Accepted 15th September 2011

DOI: 10.1039/c1cc14032a

A copper(II) complex CuRBT with a ring-closed rhodamine-

containing tripodal ligand was synthesized as a highly selective

fluorescent imaging agent for nitric oxide (NO). It featured a

700-fold fluorescent enhancement toward NO from a dark-

background with the detection limit of NO about 1 nM in

aqueous solution and could be applied for monitoring

intracellular NO.

Nitric oxide (NO) has captured great attention of biologists,

chemists, and medical researchers, since NO is an important

signaling molecule involved in the regulation of a wide range

of physiological and pathophysiological mechanisms.1

Particularly, the detection of NO in real time and in vivo and the

investigation of the roles of this signaling agent in organisms

are critical to the studies of biological functions of NO.2

It continues to be an urgent but challenging task, because of

the large diffusivity and high reactivity of NO with various

reactive oxygen species (ROS) to form reactive nitrogen

species (RNS).3 Generally, fluorescent indicators containing

biofunctional species that allow bioimaging with high spatial

and temporal resolution microscopy techniques in conjunction

with microscopy are promising techniques for elucidation of

biological functions of NO.4

Examples of the commonly used, current generation of NO

probes involve the use of o-diamino aromatics under aerobic

conditions.5 These species react with NO+ or N2O3 to furnish

fluorescent triazole derivatives and are designed as water

soluble, cell permeable and visible or longer wavelength

excitation probes for bioimaging NO.6 Transition metal com-

plexes incorporating fluorophore as part of the ligand have

been performed as platforms for NO detection7 and provide

an opportunity to explore reversible and direct sensing, for

metals can interact reversibly with nitric oxide.8 Sensors of this

class permit identification of NO from both inducible and

constitutive forms of NO synthases and facilitate investigation

of different NO functions in response to external stimuli.

Because of the large molar extinction coefficient and the

high fluorescence quantum yield, rhodamine-based dyes have

been used as effective dual responsive optical probes via

chromogenical and fluorogenical signals.9 By incorporating a

tris(2-aminoethyl)amine(tren) moiety as the efficient Cu2+

chelator to a rhodamine B group, compound RBT and its

rhodamine/dansyl derivatives have been reported10 as Cu2+-

specific chemosensors through the turn-on manner and the

FRET OFF–ON manner. Herein, we use the Cu2+ complex

CuRBT as a new and practical luminescence chemosensor for

the detection of NO in aqueous solution and in living systems.

Compound RBT was synthesized from the reaction of

tris(2-aminoethyl)amine and rhodamine B according to the

literature method.10 Copper complex CuRBT was synthesized

by refluxing a methanol solution of RBT (0.1 mmol) and

Cu(ClO4)2�6H2O (0.12 mmol) in the presence of NaCl

(0.1 mmol) for 5 h. Single crystal X-ray structure analysis

reveals that the cationic coordinated species comprises of one

Cu2+ center, one RBT ligand and one chlorine atom, with one

perchlorate anion presenting to maintain the neutrality

(Fig. 1). The Cu(II) center is surrounded by three nitrogen

donors from the RBT ligand and one chloride anion in a

planar square geometry with the deviation from the best plane

of 0.07 A. The absence of any donors in the axial position

provides much convenience for NO coordinating in the empty

positions to form [CuII–NO] species, which is anticipated as

the intermediate of the reduction and the nitrosylation process.7d

It is expected that the flexibility of the tris(2-aminoethyl)

amine unit is beneficial to the modification of the coordination

geometry suitable for Cu(I) species, which is anticipated as the

product of the reaction with NO. Cyclic voltammetric studies

of complex CuRBT in aqueous solution for the Cu(II)/Cu(I)

Scheme 1 Proposed mechanism of the reaction of CuRBT and NO.

State Key Laboratory of Fine Chemicals, Dalian University ofTechnology, Dalian, 116024, China. E-mail: hecheng@dlut.edu.cnw Electronic supplementary information (ESI) available: Experimentaldetails and additional spectroscopic data. CCDC reference number833164. For ESI and crystallographic data in CIF or other electronicformat see DOI: 10.1039/c1cc14032a

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11508 Chem. Commun., 2011, 47, 11507–11509 This journal is c The Royal Society of Chemistry 2011

couple reveal the reduction potential vs. SCE of �0.21 V

(Fig. S6, ESIw), confirming that CuRBT is indeed more easily

reduced than most of the other Cu(II)-based NO triggers.7b,12

The spirolactam form of compound RBT absorbs the UV

light and shows a band around 250 nm, thus it is colorless and

nonfluorescent. Upon addition of Cu2+, the absorbance at

238 nm decreased gradually. Detailed absorption titration

demonstrated the formation of 1 : 1 complexation species13

in solution with the dissociation constant calculated as 1.1 mM.

The ESI-MS spectrum of the titration solution exhibited an

intense peak at m/z 668.41 (Fig. S8, ESIw), which was assigned

to [(CuRBT)Cl]+ species, confirming the formation of a 1 : 1

complexation species in solution and agreeing well with the

crystal structural analysis. Importantly, single crystal X-ray

structural investigation clearly suggests the existence of the

spirolactam form of the RBTmoiety in the solid state. The two

aromatic planes of the rhodamine moiety are almost perpendi-

cular to each other with the dihedral angle of about 911. Such

a special spirolactam-ring tautomeric form of RBT inhibits the

typical emission around 580 nm (excitation at 510 nm) of

rhodamine B within a relatively wide pH range. Thus, it is

expected that the emission of the RBT moiety in copper(II)

complex will be triggered and turns on when it reacts with

nitric oxide.

A phosphate buffer solution (0.1 M, pH = 7.4) of CuRBT

thus was selected for the spectral investigation (Fig. 2). Free

CuRBT exhibited a broad but weak absorption assigned

possibly to the MLCT or d–d bands in the visible wavelength

range and showed hardly observed fluorescence (excited at 510 nm).

As an aliquot of NO stock solution was added, an absorption

of the peak around 560 nm was significantly enhanced with

log e = 3.16, suggesting the formation of the ring-opened

tautomer of CuRBT upon NO bonding. In this case, the

solution exhibited an obvious and characteristic color change

from blue to red. CuRBT thus could be used as a ‘‘naked-eye’’

detector of NO in aqueous solution.

Concomitantly, a characteristic rhodamine B emission band

centred at 580 nm (with the excitation at 510 nm) appeared

from a dark background upon the addition of NO in the

above-mentioned solution. A fluorescent enhancement of over

700-fold was observed with the quantum yield up to 0.13,

which was comparable to the 1500-fold enhancement of

AZO550 (quantum yield 0.11).14 The dark background from

which a bright and high quantum yield signal appears in

response to NO would benefit the NO imaging as exemplified

below. To identify the species responsible for NO sensing, the

luminescence of the Cu(II)-free RBT solution was monitored

by treating with excess NO. None of UV-vis variations and

fluorescence enhancement was observed in the reaction of

RBT with NO (Fig. S2 and S3, ESIw). Moreover, almost no

fluorescence increase was observed upon addition of excess

NO to a CuRBT solution containing a Cu(II) chelator

N,N0-ethanediylbis(N-carboxymethyl) glycine (Fig. S4, ESIw).These observations demonstrate that CuRBT, not RBT or Cu(II)

ion alone, is the nitric oxide indicator with fluorescence turn-on.

Under optimized conditions, the fluorescence intensity of

the probe solution is nearly proportional to the NO concen-

tration, and the purging of 1 nM NO causes more than 20%

fluorescent enhancement within 5 minutes at 298 K (Fig. 3,

inset). The detection limit of 1 nM is lower than that of most

sensitive NO sensors reported,6c,15 benefiting for the application

in biology science. To further evaluate the reaction specificity

of CuRBT with NO under physiological conditions, we

screened a wide array of possible competitive reactive oxygen

and nitrogen species and other analytes at up to 100-fold

excess. As depicted in Fig. 3, no detectable fluorescence

responses appeared upon addition of 100 equiv. of ClO�,

NO2�, NO3

�, H2O2, ONOO� and 1O2, whereas the lumines-

cence intensity was increased significantly after treatment with

NO solution, demonstrating that the fluorescence response of

the CuRBT complex is specific for NO. Furthermore, our

system is able to respond to NO in a pH range from 6.5 to 9.0,

with the fluorescence varying less than 10%, facilitating the

detection of NO in aqueous solution at the physiological

pH value.

Importantly, CuRBT exhibited the practical applicability as

a NO probe in the fluorescent imaging of living cells. MCF-7

cells were incubated with 40 mM CuRBT for 30 minutes at

room temperature to allow the probe to permeate into the

cells. When exciting at 488 nm, cells showed no intracellular

fluorescence (Fig. 4A). The cells stained with solution containing

the probes were washed three times with PBS, and then

incubated with NO solution for another 15 minutes, a significant

fluorescence emission in live cells was observed (Fig. 4B).

Bright-field measurement confirmed that the cells were viable

throughout the imaging experiments after treatment with the

probes and NO solution (Fig. 4C). As shown in Fig. 4D, the

overlay of fluorescence and bright-field images revealed that

Fig. 1 Molecular structure of CuRBT, showing the spirolactam-ring

tautomeric form of the rhodamine B moiety. Anion is omitted for

clarity. Selected bond lengths (A): Cu(1)–N(1) 1.958(4), Cu(1)–N(3)

1.984(4), Cu(1)–N(2) 2.063(3) and Cu(1)–Cl(1) 2.254(1).

Fig. 2 Absorbance spectra (left) and fluorescence responses (right) of

complex CuRBT (100 mM for UV-vis and 10 mM for fluorescence) in

the absence and presence of NO (0–10 min) in 0.1 M phosphate buffer

solution (pH = 7.4). Excitation at 510 nm.

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the fluorescence signals were localized in the perinuclear region

of the cytosol.

Electron paramagnetic resonance (EPR) spectra of CuRBT

exhibited the characteristic four-line planar pattern of Cu(II)

with the g> = 2.0127 and gJ = 2.2491 at 100 K, which was

consistent with the square planar geometry of the Cu2+

coordinated geometry.11b The addition of NO led to the

EPR-silent of CuRBT in the solution (Fig. S9, ESIw), demon-

strating the possibility of the NO-induced reduction of Cu(II)

to Cu(I) during the turn-on fluorescence response of NO,

forming NO+. While the addition of [Cu(CH3CN)4](ClO4)

to the aqueous solution of RBT was not found to change the

fluorescence (Fig. S3, ESIw). This result indicates that

reduction of Cu(II) to Cu(I) alone does not cause fluorescence

in the NO capture, which differs from prior observations for a

Cu(II)-based system.7b,f In comparison with the other reported

results, one mechanism could be supposed involving the initial

NO coordination to the square planar copper ion followed by

formation of NO+ through the NO-induced reduction of

Cu(II) to Cu(I), then NO+ migrated to the amide nitrogen to

open the spirolactam, resulting in concomitant nitrosation and

turn-on fluorescence response. LC-MS of the reaction mixture

exhibited a new peak at m/z 601.3, assignable to the species of

[(RBT–NO) + H]+ (Fig. S10, ESIw). This result provides thepossibility of nitrosation of RBT to generate RBT–NO. The

IR spectrum of the reaction mixture containing CuRBT and

NO exhibited a new vibration band at 1384 cm�1, corres-

ponding to nNN–O,3b giving further support to the formation of

RBT–NO in the solution.

Notes and references

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8 P. C. Ford and I. M. Lorkovic, Chem. Rev., 2002, 102, 993.9 H. N. Kim, M. H. Lee, H. J. Kim, J. S. Kim and J. Y. Yoon,Chem.Soc. Rev., 2008, 37, 1465.

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11 (a) P. G. Gunasekar, A. G. Kanthasamy, J. L. Borowitz andG. E. Isom, J. Neurosci. Methods, 1995, 61, 15; (b) L. E. McQuade,M. D. Pluth and S. J. Lippard, Inorg. Chem., 2010, 49, 8025.

12 L. Prodi, M. Montalti, N. Zaccheroni, F. Dallavalle, G. Folesani,M. Lanfranchi, R. Corradini, S. Pagliari and R. Marchelli, Helv.Chim. Acta, 2001, 84, 690.

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Fig. 4 Confocal fluorescence images of MCF-7 cells. (A) Cells

incubated with 40 mM CuRBT for 30 min. (B) Cells incubated with

the above-mentioned solution for 30 min, then washed three times,

and further stained with NO solution for 15 min. (C) Bright-field

image of cells showed in panel (B). The overlay image of (B) and (C) is

shown in (D) (lex=488 nm).

Fig. 3 Fluorescence responses of CuRBT (10 mM) upon addition of

100 equiv. of ROS and RNS for 2 h in phosphate buffer solution

(0.1 M, pH = 7.4). Inset: fluorescence intensity changes of CuRBT

(10 mM) in aqueous solution upon addition of NO solution (1–10 nM).

The intensities were recorded at 580 nm and normalized with respect

to the emission of CuRBT. Excitation was provided at 510 nm.

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