This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 5431–5433 5431

Cite this: Chem. Commun., 2011, 47, 5431–5433

Selective and sensitive ‘‘turn-on’’ fluorescent Zn2+ sensors based

on di- and tripyrrins with readily modulated emission wavelengthsw

Yubin Ding,aYongshu Xie,*

aXin Li,

aJonathan P. Hill,*

bWeibing Zhang

aand

Weihong Zhua

Received 15th March 2011, Accepted 24th March 2011

DOI: 10.1039/c1cc11493j

Di- and tripyrrin sensors D1–D4 exhibit CHEF-type fluorescence

enhancement by factors up to 72 upon addition of 1 equiv. Zn2+

,

with tunable emission colours between green (D1) and red (D4).

As the second most abundant transition metal in the human

body after iron, zinc plays vital roles in numerous biological

processes, including brain activity, gene transcription, and

immune function.1 Therefore, understanding the distribution

and biochemical action of Zn2+ in living tissues is a subject of

great importance.1,2 Thus, Zn2+ sensing has attracted increasing

interest in the chemical and biological sciences.

Fluorescent sensors can be implemented using simple protocols

and with high sensitivity, which have made them promising

candidates for cation sensing.3 In bioimaging, the employment

of long wavelength emission may reduce autofluorescence

interference and photodamage to living cells. The most common

types of fluorescent sensors are based on photoinduced

electron-transfer (PET) or intramolecular charge transfer

(ICT) mechanisms,4 and they have been extensively investi-

gated leading to successful applications in imaging of Zn2+ in

living cells.4d,5 However, these sensors usually require multi-

step syntheses involving severe reaction conditions and expensive

chemicals such as palladium catalysts.

On the other hand, sensors based on the chelation-enhanced

fluorescence (CHEF) effect are relatively simple to prepare,

since they require only a single chromophore containing the

necessary chelating atoms. So far, a number of such sensors

have been reported,6 but ‘‘turn-on’’ type Zn2+ CHEF sensors

that emit at wavelengths longer than 600 nm are still relatively

rare.6c,e Dipyrrins are dipyrrolic compounds exhibit weak

fluorescence, although some of their Zn2+ and other metal

complexes do exhibit strong emission.7 This implies that they

could be developed as ‘‘turn-on’’ fluorescent Zn2+ sensors.

However, until now little effort has been made to determine

their utility despite the fact that various porphyrinoids with

more complicated structures have been designed for Zn2+

sensing.8

Based on these facts and our previous work on related

systems,9 here we report four readily synthesized ‘‘turn-on’’

fluorescent Zn2+ sensors (D1–D4) based on dipyrrins and

tripyrrins. Upon addition of 1 equiv. Zn2+, their fluorescence

can be enhanced by factors up to 72, with the emission colours

varying from green to red. Each of the sensors shows good

sensitivity and selectivity for Zn2+. Furthermore, D4 was

successfully applied for sensing in aqueous media and living

cells, indicative of a promising fluorescent Zn2+ chemosensor

in certain practical applications.

D1–D4 were easily synthesized by simple oxidation of

the corresponding dipyrromethanes or tripyrrane and fully

characterized by 1H NMR, 13C NMR, and HRMS (Scheme 1

and S1w, Fig. S1–S6w).10 Upon addition of Zn2+ to DMF

solutions of these sensors, a strong fluorescence enhancement

was easily apparent even to the naked eye (Fig. S11c–dw). Toinvestigate the sensing behaviour in detail, the UV-Vis spectral

changes of the sensors upon addition of Zn2+ were measured.

For D4 for example, the titration of Zn2+ to its DMF solution

induced a colour change from red to blue (Fig. S11a–bw), andthe peak centered at 532 nm in its UV-vis spectrum (Fig. 1a)

gradually decreased, with the concurrent appearance of a new

band at 624 nm. Similar absorption changes were observed for

D1, D2 and D3 (Fig. S8a–S10aw). These spectral changes canbe attributed to the formation of zinc complexes of the

sensors. As expected, while sensors D1–D4 exhibit rather

weak emission, their fluorescence intensity was significantly

enhanced upon addition of Zn2+ (Fig. 1b, S8b–S10bw). Notably,

a most pronounced 72-fold enhancement for D4 was observed

(Fig. 1b). The fluorescence enhancement can be ascribed to the

CHEF effect associated with better rigidity and planarity of

the sensor molecules induced by chelation of Zn2+, which is

supported by the respective crystal structures (vide infra).

Scheme 1 Chemical structures of chemosensors D1, D2, D3 and D4.

a Key Laboratory for Advanced Materials and Institute of FineChemicals, East China University of Science and Technology,Shanghai, P. R. China. E-mail: yshxie@ecust.edu.cn;Fax: (+86) 21-6425-2758; Tel: (+86) 21-6425-0772

bWPI-Center for Materials Nanoarchitectonics, National Institute forMaterials Science (NIMS), Namiki 1-1, Tsukuba, Ibaraki, Japan

w Electronic supplementary information (ESI) available: Full experimentaland crystallographic data and Fig. S1 to S18. CCDC 808307–808309. ForESI and crystallographic data in CIF or other electronic format see DOI:10.1039/c1cc11493j

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5432 Chem. Commun., 2011, 47, 5431–5433 This journal is c The Royal Society of Chemistry 2011

Moreover, in the presence of Zn2+, emission colours can be

readily modulated from green (D1) to red (D4) (Fig. 2, S11dw),which is critically dependent on the size of the p-electronconjugation system. Compared with the parent dipyrrin of D1,

sensor D2 has one additional formyl group, D3 has one

additional benzoyl group, and D4 has one more pyrrolic group

linked by methylene to the dipyrrin unit. From D1 to D4, the

HOMO–LUMO energy gaps of the corresponding Zn complexes

are decreased successively from 3.11 to 2.36 eV (Table S2w,Fig. S14w), resulting in a successive increase of the emission

maxima wavelengths from 514 to 637 nm. Apparently, the

modification at the pyrrolic a-position in the dipyrrin unit can

realize a convenient way to tune the emission colours.

To further understand the molecular structures of the zinc

complexes, single crystals of [Zn(D2)2], [Zn(D3)2]�MeOH and

[ZnD4(H2O)2]�2MeOH were grown and analyzed by X-ray

diffraction. The Zn2+ binding modes of 1 : 2 for D2 and

D3, and 1 : 1 for D4 are observed in the crystal structures

(Fig. 3, S12, S13w), which are consistent with those obtained

from the absorption and fluorescence measurements (vide supra).

In the complexes, each of the coordinating dipyrrin or tripyrrin

ligands is nearly planar. In the case of [ZnD4(H2O)2]�2MeOH,

the dihedral angles between pyrrolic units are significantly

smaller (between 3.981 and 8.661) than those observed in

the crystal of free D4 ligand10 (between 9.971 and 26.121; see

Table S1w). Consequently, Zn2+ chelation can induce better

planarity and rigidity of the sensor molecules, and suppress

intramolecular distortions from planarity, resulting in the

above-mentioned CHEF effect.

One of the most important parameters in cation sensing is

the detection limit. For many practical purposes, it is impor-

tant to sense cations at extremely low concentrations. Thus,

based on the fluorescence titration measurements,11 detection

limits of D1, D2, D3 and D4 for Zn2+ were found to be 1.1 �10�7, 2.7 � 10�7, 1.3 � 10�7 and 4.6 � 10�8 M (Fig. S15–18w),respectively. Impressively, D4 can be applied for detection of

Zn2+ at concentrations as low as 10�8 M, which might fully

meet the requirements in biosensing.

Selectivity is another major issue in the field of cation

sensing. All the sensors show good selectivity for Zn2+

(Fig. 4a, S7c–S10cw). As a case of D4 in DMF or in HEPES

buffer (pH 7.2) solution, when 1 equiv. of cation (for Na+,

K+, 100 equiv. and Ca2+, 10 equiv.) was added, only Zn2+

can significantly enhance the fluorescence, whereas, other

divalent metals and in vivo abundant alkaline and alkaline

earth metal cations only induce negligible fluorescence changes

or even quenching. It is well known that Zn2+ fluorescent

sensors may be detrimentally affected by interference from

other cations, especially Cd2+ or Cu2+.9a,12 Thus, competition

experiments were carried out to further elucidate cation

selectivity. In the solutions of the sensors, the addition of

competing cations did not interfere significantly with Zn2+

sensing (Fig. 4b, S7d–S10dw). Accordingly, sensors D1–D4

can be established as a novel and promising type of highly

sensitive and selective ‘‘turn-on’’ fluorescent Zn2+ sensors.

For the fluorescent sensing of Zn2+ by D4, the emission

peak is centered at a long wavelength of 637 nm, indicative of

the potential application in bioimaging. To this end, we

employed D4 to image low concentrations of Zn2+ in KB

cells (human nasopharyngeal epidermal carcinoma cell) with

the use of a confocal fluorescence microscopy.13 Bright-

field measurements confirmed that the cells treated with

Zn2+ and D4 were viable throughout the imaging experiments

(Fig. 5a and d). In the control experiment, the staining of KB

cells with D4 led to weak intracellular fluorescence (Fig. 5b).

In contrast, a significant increase in fluorescence from the

Fig. 1 (a) UV-vis spectral changes during the titration of D4 (10 mM)

with Zn2+ (0–2.0 equiv.) in DMF. (b) Corresponding fluorescence

emission spectral changes with lex fixed at 568 nm (one of the isosbestic

points).

Fig. 2 Fluorescence responses of sensors D1, D2, D3 and D4 (10 mM)

in the presence of 1 equiv. of Zn2+ in DMF.

Fig. 3 X-ray crystal structure of [ZnD4(H2O)2]�2MeOH complex.

(a) Top view, (b) side view. Hydrogen atoms are omitted for clarity.

Fig. 4 Relative fluorescence intensity of 10 mM D4 in DMF upon

excitation at 568 nm (one of the isosbestic points): (a) in the presence

of various metal ions. (b) White bars represent the addition of 1 equiv.

of metal ions (for Na+, K+, 100 equiv. and Ca2+, 10 equiv.). Black

bars represent the addition of 1 equiv. of Zn2+ mixed with 1 equiv. of

indicated metal ions (for Na+, K+, 100 equiv. and Ca2+, 10 equiv).

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This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 5431–5433 5433

intracellular area was observed when the cells were treated with

Zn(OAc)2 in the growth medium and then with D4 (Fig. 5e).

The overlay of fluorescence and bright-field images reveals that

the fluorescence signals are localized in the perinuclear area of

cytosols, indicating a subcellular distribution of Zn2+ and good

cell-membrane permeability of D4 (Fig. 5c and f), with the

practical applicability for Zn2+ imaging in living cells.

In conclusion, we report four easily synthesized highly sensitive

and selective ‘‘turn-on’’ fluorescent sensors for Zn2+ based on

the CHEF mechanism. Upon addition of 1 equiv. Zn2+, the

sensors exhibit fluorescence enhancement by factors up to 72,

with the emission wavelength easily modulated between 514

and 637 nm simply by varying the sensor structure. D4 was

successfully applied in both HEPES buffer solution and for the

imaging of Zn2+ in living cells with several advantages such

as cell-permeability, the desired long emission wavelength

beneficial for deep light penetration and weak autofluorescence

of biological tissues, a CHEF-based turn-on fluorescence type

to get the maximum signal-to-noise ratio, and being capable

of discriminating Zn2+ from other cations, especially with

little interference of Cd2+ and Cu2+. These sensors may be

further developed as a novel type of readily synthesized, high

performance fluorescent Zn2+ sensor with the practical

applicability for Zn2+ imaging in living cells and Zn2+ sensing

in relevant aqueous systems.

This work was financially supported by NSFC, Innovation

Program of Shanghai Municipal Education Commission, the

Fundamental Research Funds for the Central Universities

(WK1013002), SRFDP (200802510011 and 20100074110015),

the Oriental Scholarship, and National Basic Research 973

Program (2011CB910404).

Notes and references

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10 D1 and D4 were prepared acording to the procedures reported inthe following papers: (a) J. Y. Shin, S. S. Hepperle, B. O. Patrickand D. Dolphin, Chem. Commun., 2009, 2323; (b) J. Y. Shin,D. Dolphin and B. O. Patrick, Cryst. Growth Des., 2004, 4, 659.

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Fig. 5 Confocal fluorescence and bright field images of KB cells:

(a)–(c) cells incubated with D4 (10 mM) for 0.5 h at 37 1C. (d)–(f) Cells

pretreated with Zn(AcO)2 (20 mM) for 0.5 h then incubated with D4

(10 mM) for 0.5 h. (a) and (d): Bright field, (b) and (e): fluorescence,

and (c) and (f): overlay.

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