This journal is c The Royal Society of Chemistry 2012 Chem. Commun.

Cite this: DOI: 10.1039/c2cc35967g

Ratiometric fluorescence chemodosimeters for fluoride anion based on

pyrene excimer/monomer transformationw

Lizhi Gai,aHuachao Chen,

bBin Zou,

aHua Lu,*

aGaoqiao Lai,

aZhifang Li*

aand

Zhen Shen*b

Received 17th August 2012, Accepted 17th September 2012

DOI: 10.1039/c2cc35967g

Two pyrene dimers containing an –O–Si–Si–O� or �O–Si–O�linkage have been designed which exhibit ratiometric excimer/

monomer emission upon fluoride anion induced Si–O bond cleavage.

Incorporation of the probe into water soluble polymeric nanoparticles

enhances its intracellular uptake and displays ratiometric fluorescent

sensing for F� in living cells.

The search for the recognition and sensing of biologically important

anions has emerged as a research area of considerable importance.1

Among various anions, the fluoride anion (F�) is one of the most

significant anions due to its mild toxicity and important role in

bone-growth.2 A number of chromogenic and fluorogenic

chemosensors for F� have been designed and characterized,

most of them are based on the hydrogen bonding or Lewis acid

coordination, however, it has been proved to be very difficult to

detect F� in aqueous solution.3 The chemodosimeter approach

based on the extraordinary affinity between fluoride and silicon,

which showed fast response with high selectivity and sensitivity

in aqueous systems, has received increasing attention.4 Ratio-

metric measurements that use one excitation wavelength and

take the intensity ratio at two different absorption or emission

wavelengths can offer intrinsic advantages in both chemical and

biological sensing systems.5 Although a few ratiometric fluorescent

chemodosimeters for fluoride anion in aqueous systems have

been reported,6 there is only one example of the ratiometric

chemodosimeter to monitor fluoride anion in living cells.6c

As popular fluorophores, pyrene-labeled molecules have many

advantages such as the ratio between the emission intensities of

the monomer and excimer, relatively large fluorescence quantum

yield and long lifetimes.7 An excimer can be formed when an

excited-state pyrene molecule is brought into close proximity

with a ground state pyrene moiety.8 The formation of the

excimer results in a bathchromic shift of the fluorescent wave-

length (from 375 and 398 nm for the monomer) to a longer

wavelength (470–490 nm).9 On the basis of the intensity ratio of

the monomer to the excimer peaks, signal fluctuations can be

cancelled, and the impact of environmental quenching can be

minimized.10 Research in this area has been successful in the

selective detection of ions, small molecules and DNA.11 However,

the majority of ratiometric sensors are based on a covalent linked

three component system containing a receptor, a transducer and a

fluorophore. Moreover, their sensing mechanism is based on the

complexation or hydrogen bonding interaction with the analyte.

In this communication, two pyrene moieties linked with a

flexible�O–Si–Si–O– or�O–Si–O– chain which show excimer

emission of pyrene have been designed. The Si–O bond is

cleaved specifically by the fluoride anion leading to pyrene

monomer emission (Fig. 1). Furthermore, F� sensing perfor-

mance and intracellular uptake of the probe incorporated into

nanoparticles have been evaluated by confocal laser scanning

microscopy (CLSM) in living HeLa cells. To the best of our

knowledge, this is the first example of a chemodosimeter based

on the pyrene excimer/monomer transformation mechanism.

As shown in Scheme 1, the pyrene dimers 1 and 2 and the

reference pyrene monomer 3 were prepared from the reaction

of 1-hydroxymethylpyrene with 1,2-dichlorotetramethyldisilane,

dichlorodimethylsilane or chlorotrimethylsilane, respectively, in

THF containing Et3N in high yields (the synthetic procedures are

provided in full in ESIw). The 1H NMR spectra showed that the

proton signals of the pyrene rings and methyl groups in the dimers

1 and 2 are high-field shifted compared with those in the monomer

3 (Fig. S1, ESIw), this might be due to the ring-current effects of the

bipyrene units that influence each other, resulting in the shielding of

the proton chemical shifts from each pyrene ring.12

Fig. 1 Ratiometric fluorescent probe for F� based on pyrene dimers

as chemodosimeters.

a Key Laboratory of Organosilicon Chemistry and MaterialTechnology, Ministry of Education, Hangzhou Normal University,Hangzhou, 310012, P. R. China. E-mail: hualu@hznu.edu.cn,zhifanglee@hznu.edu.cn; Fax: +86 (0)571-28868529;Tel: +86 (0)571-28867825

b State Key Laboratory of Coordination Chemistry, Nanjing NationalLaboratory of Microstructures, Nanjing University, Nanjing, 210093,P. R. China. E-mail: zshen@nju.edu.cn; Fax: +86 (0)25-83314502;Tel: +86 (0)25-83686679

w Electronic supplementary information (ESI) available: Experimentaldetails, additional spectroscopic properties and NMR spectra. SeeDOI: 10.1039/c2cc35967g

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Optimized geometries of 1 and 2 obviously show p–p stacking

interactions between the two pyrene moieties, in which the

pyrene units are separated by about 7 A and 5 A for 1 and 2,

respectively (Fig. 2). The p–p interaction in 2 is deduced to be

stronger than that in 1 owing to the shorter dimeric distance.

As shown in Fig. 3a, the fluorescence spectra of compounds

1 and 2 exhibit weak monomer emission together with a

strong, broad and featureless band typical for the pyrene

excimer emission in the range of 470 to 480 nm. The excimer

emission band of 2 is slightly redshifted compared to that of 1,

most likely due to the enhanced interaction between the two pyrene

units through a shorter chain linkage. Compound 3 exhibits two

strong peaks at 378 nm and 396 nm with well-resolved vibronic

structure, which is typical of the emission band of the pyrene

monomer. There is almost no emission band beyond 430 nm,

indicating the absence of excimer formation of 3 in the solution.

Compared to the emission spectrum of 3, the excimer emission

of 1 and 2 can be attributed to the intramolecular interaction

mechanism of pyrene units, rather than an intermolecular one.

The anion binding properties of compounds 1–3 were

investigated by monitoring the changes in fluorescence and

UV-vis absorption spectra. All the titration experiments were

carried out in THF–H2O (v/v, 50/50) by adding different

anions. Upon addition of 10 equiv. of F�, Cl�, Br�, I�,

CO32�, NO2

�, NO3�, SCN�, ClO4

�, SO42�, HPO4

2�, and

H2PO4� into a solution of 1 (10 mM), a significant quenching

in excimer emission and a large increase in the monomer

emission are observed for F�. Little or no change was

observed in the fluorescence spectrum of 1 in the presence of

other anions (Fig. 4 and Fig. S2, ESIw). This is attributed to

the specific cleavage of Si–O bonds by F� leading to the

breakage of the stacked conformation of two pyrene moieties.

The cleavage of Si–O bonds was confirmed by the mass

spectrum of 1 with F� (Fig. S9, ESIw). The 1H NMR spectrum

of 1 in the presence of fluoride anion in CDCl3 was measured.

After the addition of F�, most of the protons of pyrene rings

shift downfield similar to those of 3, suggesting the breakage

of the stacking-pyrene ring current (Fig. S10, ESIw).12 The

monomer/excimer emission ratio of 1 responds proportionally

to the concentration of F� (Fig. 3b). The limitation for the

detection of F� is in the micromolar range. Therefore, probe 1

is a highly selective and sensitive ratiometric chemodosimeter

for F� in aqueous solution. Furthermore, the absorption

maximum of 1 is hypsochromically shifted 2 nm after addition

of fluoride anion, which indicates that the two pyrene moieties

have some interaction in the ground state (Fig. S8, ESIw).12 Thespectroscopic response of bipyrene 2 toward various anions

shows similar behavior to that of 1 (Fig. 4 and Fig. S3, ESIw).Unlike bipyrene compounds, monopyrene 3 shows no specific

spectroscopic change to most of the anions (Fig. S4, ESIw).The sensing properties of probe 1 in living HeLa cells were

also investigated. Biodegradable poly(D,L-lactic acid) (PLA)

nanoparticles were selected as a matrix for loading and sub-

sequent controlled release of hydrophobic probe 1 into cells by

Scheme 1 Syntheses of pyrene derivatives 1, 2 and 3.

Fig. 2 Optimized geometries of 1 (left) and 2 (right) using a B3LYP/

6-31G(d) method.

Fig. 3 (a) Fluorescence spectra of 10 mM 1, 2 and 3 in THF/H2O

(v/v, 50/50) solution. Inset: picture of the solutions excited under the UV

light at 365 nm. (b) Fluorescent titration spectra of 10 mM 1 in THF/H2O

(v/v, 50/50) in the presence of increasing amount of F� anion. Inset:

titration curve of I378 nm/I470 nm vs. [F�]/[1] (lex = 335 nm).

Fig. 4 Fluorescence response of 10 mM 1 and 2 toward various

anions in THF/H2O (v/v, 50/50), color bars represent the fluorescence

intensity ratio I378/I470 upon the addition of 10 equivalents of various

anions.

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This journal is c The Royal Society of Chemistry 2012 Chem. Commun.

using an oil-in-water (O/W) emulsion and a subsequent solvent

evaporation method.13 The F� sensing performance and intra-

cellular uptake of the nanoparticles containing probe 1 were

evaluated by CLSM in living HeLa cells. HeLa cells incubated

with 1–PLA nanoparticles displayed marked intracellular pyrene

excimer emission at 440–600 nm (Fig. 5a–c). When the above

HeLa cells were treated with 100 mM F� at 37 1C for 2 h, they

clearly showed intracellular pyrene monomer luminescence at

shorter wavelengths in the range of 410–440 nm (Fig. 5e–g).

The marked change observed in intracellular fluorescence was

attributed to the reaction between probe 1 and F�. Bright field

measurements confirmed that the cells were viable throughout

the imaging experiments (Fig. 5d and h). These results demon-

strated that chemodosimeter 1 can be used for the ratiometric

fluorescence imaging of F� in living cells.

In conclusion, ratiometric fluorescent chemodosimeters 1 and 2

based on pyrene dimers linked through –O–Si–Si–O� or

�O–Si–O� have been designed and characterized, which exhibit

high selectivity toward F� over various anions and biorelevant

analytes, owing to the strong affinity of F� toward silicon.

Moreover, confocal fluorescence microscopy experiments have

established the utility of water soluble nanoparticles incorporated

with 1 for monitoring fluoride anion with ratiometric fluorescence

in living cells. The pyrene monomer 3 shows almost no spectro-

scopic change upon addition of various anions, indicating that the

idea that two chromophores linked by a flexible chain containing

reactive site is inspiring. Considering the diversity of reaction

sites and chromophores, the search for new chromophores and

reactions for detecting biologically important small molecules,

ions, and DNA is underway.

Financial support from the National Natural Science Foun-

dation of China (nos. 21101049, 20971066 and 21021062) and

the Major State Basic Research Development Program of

China (Grant No. 2011CB808704) is gratefully acknowledged.

Notes and references

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Fig. 5 Confocal fluorescence images of live HeLa cells: the cells were

incubated with probe 1 in PLA nanoparticles (0.25 mg mL�1) for 4 h;

(a) fluorescence images with emission collected at 410–440 nm by the

blue channel, (b) green channel at 440–600 nm, (c) overlaid images of

panels a and b, (d) bright-field transmission image. The above cells

after addition of 100 mM F� for 2 h, (e) fluorescence images with

emission collected at 410–440 nm by the blue channel, (f) green

channel at 440–600 nm, (g) overlaid images of panels e and f, (h)

bright-field transmission image.

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