Reactive fluorescence turn-on probes for fluoride ions in purely aqueous media fabricated from functionalized responsive block copolymers

Article abstract of DOI:10.1021/ma2018588

We report on the fabrication of a novel type of responsive double hydrophilic block copolymer (DHBC)-based highly selective and sensitive fluorescence "turn-on" reactive probes for fluoride ions (F ^-) working in purely aqueous media by exploiti

Full text of DOI:10.1021/ma2018588

ARTICLE
pubs.acs.org/Macromolecules
Reactive Fluorescence Turn-On Probes for Fluoride Ions in Purely
Aqueous Media Fabricated from Functionalized Responsive Block
Copolymers
Yanyan Jiang,† Xianglong Hu,† Jinming Hu,† Hao Liu,† Hui Zhong,*,‡ and Shiyong Liu*,†
†CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, Hefei National Laboratory for
Physical Sciences at the Microscale, University of Science and Technology of China, Hefei, Anhui 230026, China
‡School of Chemistry and Chemical Engineering, Huaiyin Normal University, Huaiyin, Jiangsu 223300, China
S Supporting Information
b
ABSTRACT: We report on the fabrication of a novel type of
responsive double hydrophilic block copolymer (DHBC)-based
highly selective and sensitive fluorescence “turn-on” reactive probes
for fluoride ions (Fꢀ) working in purely aqueous media by exploiting
Fꢀ-induced cyclization reaction of nonfluorescent moieties to induce
the formation of fluorescent coumarin moieties, as inspired by the
previous work of the Swager research group (Angew. Chem. Int. Ed.
2003, 42, 4803). Diblock copolymers bearing Fꢀ-reactive moieties
(SiCouMA) in the thermoresponsive block, PEO-b-P(MEO2MA-
co-OEGMA-co-SiCouMA), were synthesized at first via reversible
additionꢀfragmentation chain transfer (RAFT) technique followed
by postmodification, where PEO, MEO2MA, and OEGMA are
poly(ethylene glycol), di(ethylene glycol) monomethyl ether metha-
crylate, and oligo(ethylene glycol) monomethyl ether methacrylate, respectively. As-synthesized diblock copolymers molecularly
dissolve in water at room temperature and self-assemble into micellar nanoparticles above the critical micellization temperature
(33 ꢀC). In the presence of Fꢀ ions, deprotection of nonfluorescent SiCouMA moieties followed by spontaneous cyclization
reaction leads to the formation of highly fluorescent coumarin residues (CouMA). Thus, PEO-b-P(MEO2MA-co-OEGMA-co-
SiCouMA) diblock copolymers can serve as highly efficient and selective fluorescence “turn-on” reaction probes for Fꢀ ions in
aqueous media. In the range of 0ꢀ1600 equiv of Fꢀ ions, diblock unimers and micellar solutions at 20 and 40 ꢀC exhibit ∼88-fold
and ∼30-fold increase in fluorescence emission intensity (20 min incubation time), respectively. The detection limits were
determined to be 0.065 and 0.05 ppm for diblock unimers and micelles, respectively. Most importantly, in the low Fꢀ concentration
range, excellent linear correlation between Fꢀ concentration and emission intensity was observed (0ꢀ15 ppm for unimers at 20 ꢀC
and 0ꢀ8 ppm for micelles at 40 ꢀC). Interestingly, upon complete transformation of nonfluorescent SiCouMA moieties into
fluorescent CouMA, the emission intensity of diblock copolymer solution decreases linearly with temperatures in the range of
20ꢀ60 ꢀC, suggesting its further application as fluorometric temperature sensors. To the best of our knowledge, this work
represents the first example of Fꢀ-reactive polymeric probes working in purely aqueous media, which are capable of highly sensitive
and selective fluorescent Fꢀ sensing in the form of both unimers and micellar nanoparticles.
’ INTRODUCTION
Fꢀ ions has recently emerged to be the most convenient
technique due to its simplicity, low cost, low detection limit,
and suitability for detection at the intracellular and tissue-specific
level. Moreover, concerning its practical applications, aqueous
detection media is the best choice for optical Fꢀ probes.
Up to now, three types of design strategies have been exploited
to develop optical (colorimetric and/or fluorometric) probes of
Fꢀ ions, namely, supramolecular recognition,11ꢀ25 Lewis acidꢀ
base interactions,26ꢀ32 and Fꢀ ion-induced chemical reactions.33ꢀ45
Detection systems employing the former two strategies typically
The sensing and detection of fluoride ions (Fꢀ) has aroused
considerable interest in the past decade due to that Fꢀ is highly
relevant to health care, detection of nerve gases, and refinement
of uranium resources.1ꢀ5 For drinking water quality control, the
EPA (United States Environmental Protection Agency) set a
standard of 2ꢀ4 ppm to protect against osteofluorosis and dental
fluorosis. However, for human beings of all ages, insufficient
fluoride intake will incur increased risk of dental caries.6 There-
fore, it is highly important to develop selective, sensitive, fast, and
cost-effective detection techniques for fluoride ions. Conven-
tional analytical methods for Fꢀ ions typically involve the use of
ion chromatography,7 ion-selective electrodes,8,9 and capillary
zone electrophoresis.10 In addition, the optical detection of
Received: August 14, 2011
Revised:
October 16, 2011
Published: October 25, 2011
r
2011 American Chemical Society
8780
dx.doi.org/10.1021/ma2018588 Macromolecules 2011, 44, 8780–8790
|

Macromolecules
ARTICLE
Scheme 1. Schematic Illustration for the Fabrication Responsive Double Hydrophilic Block Copolymer- (DHBC-) Based Highly
Selective and Sensitive Fluorescence Turn-On Probes for Fluoride Ions Working in Purely Aqueous Media by Exploiting Fꢀ-Induced
Cyclization Reaction of Nonfluorescent Moieties To Induce the Formation of Fluorescent Coumarin Moieties within the
Thermoresponsive Block
need to be conducted in organic solvents due to competitive
interactions with water molecules, thus Fꢀ sensing is only
suitable for tetrabutylammonium salts and not quite applicable
for the sodium or potassium salts; moreover, interference from
the presence of H2PO4ꢀ, AcOꢀ, and CNꢀ ions is unavoidable
and the detection selectivity is a severe issue. To solve the
selectivity problem, Fꢀ-induced chemical reactions such as the
quite specific and selective SiꢀO bond cleavage have been utilized
to design fluorescence “turn-on” reactive Fꢀ sensors. Numerous
fluorescent dyes such as coumarin,37,40,46 fluorescein,42 cyanine,38
and 1,8-naphthalimide derivatives35 were rendered nonfluores-
cent at first by caging with trialkylsilyoxy moiety, the presence of
Fꢀ ions can induce the deprotection of caging groups and turn-on
the fluorescence emission. Quite recently, the construction of
novel type of reactive fluorescence “turn-on” probes by employ-
ing Fꢀ ion-induced direct cyclization reaction or the polymeri-
zation of nonfluorescent precursors into fluorescent species was
also reported.47,48
Note that the above chemical reaction-based fluorometric
Fꢀ probes are exclusively based on small molecule dyes. In most
cases, the measurements need to be conducted in purely organic
solvents or less efficiently in organic solvent/water mixtures. This
poses severe limitations to their biological applications such as
bioassays at intracellular and tissue levels. Currently, there exist only
two literature reports concerning small molecule-based fluorometric
Fꢀ probes in purely aqueous media.38,49 Zhang et al.38 reported the
synthesis of tert- butyldiphenylsilyl caged dye bearing a quantized
benzothiazolium moiety, which is water- soluble and can be applied
for the intracellular fluorometric detection and imaging of fluoride
ions. Yang et al.49 constructed ratiometric fluorescent Fꢀ probes on
the basis of hydrophobic small molecule dyes embedded within
surfactant micelle cores embedded, which can also work in aqueous
media. Another notable disadvantage of small molecule reactive Fꢀ
probes is that they are difficult to be integrated with other analyte-
sensing functions. Considering potential in vivo applications, they
also possess inherent limits such as rapid elimination and extravasa-
tion out of the vasculature during blood circulation, which will
restrict the timing for measurements.
We have recently been interested in the design of detection
and sensing systems on the basis of responsive polymers and
their assemblies.50ꢀ54 The integration of responsive polymers
with small molecule sensing motifs can allow for the construction
of novel type of polymeric probes possessing multiple advantages
such as excellent water solubility, multifunctional sensing cap-
ability, tunable detection sensitivity and selectivity, and enhanced
biocompatibility. Concerning fluorescent polymeric probes for
Fꢀ ions, there exist only several relevant accounts. Tian et al.55
reported that naphthalimide-functionalized poly(phenylacetylene)
can serve as ratiometric fluorescent Fꢀ probes in MeCN. We
recently reported that secondary amine moieties in nitrobenzo-
furazan derivatives such as 4-(2-acryloyloxyethylamino)-7-nitro-
2,1,3-benzoxadiazole (NBDAE) and NBDAE-containing poly-
mers can serve as a new type of colorimetric and fluorometric Fꢀ
probes with a detection limit down to ∼0.8 μM.56 Unimers and
assembled micellar nanoparticles of coilꢀrodꢀcoil ABA triblock
copolymers consisting of a fluorescent conjugated polymer
middle block and two outer NBDAE-containing coil blocks also
exhibits ratiometric fluorescent sensing capability for Fꢀ ions in
acetone.57
The only example of polymeric fluorometric fluoride ion
sensor exhibiting Fꢀ ion-reactive characteristics was reported
in 2003 by Swager and his co-workers.58 They ingeniously
incorporated newly designed chemical reaction-based Fꢀ sensing
moieties, which are initially nonfluorescent but subjected to
spontaneous deprotection and subsequent structural rearrange-
ment into highly fluorescent coumarin derivatives in the presence
of Fꢀ ions, into the side group of fluorescent conjugated
polymer, polythiophene. Thus, Fꢀ ion-induced generation of
coumarin moieties in THF can effectively amplify the emission
response via exciton migration from the conjugated backbone,
resulting in ∼100-fold enhancement in Fꢀ detection sensitivity for
the modified conjugated polymer, as compared to the small molecule
counterpart.58,59
It should be noted that all the above four examples of
polymeric fluorescent Fꢀ probes can only be applied in organic
media. On the basis of the above analysis, it is highly desirable to
8781
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
Scheme 2. Reaction Schemes Employed for the Synthesis of Fꢀ-Reactive Small Molecule Precursor (SiCouꢀOH, 7)
develop polymers or polymeric assemblies-based reactive fluor-
escence “turn-on” Fꢀ probes that can effectively work in purely
aqueous media. Herein, we report on the fabrication of a novel
type of responsive double hydrophilic block copolymer-(DHBC-)
based highly selective and sensitive fluorescence “turn-on” re-
active probes for Fꢀ ions working in aqueous media by utilizing
Fꢀ-induced cyclization reaction of nonfluorescent moieties to
induce the formation of fluorescentcoumarin moieties (Scheme 1).
It is worthy of noting that the Fꢀ ion sensing structural motif
was inspired by the previous work of Swager research group.57
Diblock copolymers bearing Fꢀ-reactive moieties (SiCouMA) in
the thermoresponsive block, PEO-b- P(MEO2MA-co-OEGMA-
co-SiCouMA), were synthesized at first (Schemes 2 and 3),
where PEO, MEO2MA, and OEGMA are poly(ethylene
glycol), di(ethylene glycol) monomethyl ether methacrylate,
and oligo(ethylene glycol) monomethyl ether methacrylate,
respectively. The thermo-induced micellization of as-synthe-
sized SiCouMA-labeled diblock copolymer, the Fꢀ-sensing
capability including detection limits, sensitivity, and selectivity
were explored in detail for both diblock unimers and micelles
at temperatures below and above the critical micellization
temperature (CMT).
and distilled just prior to use. 4-Hydroxy-2-methoxybenzaldehyde was
recrystallized twice from deionized water and dried under reduced
pressure. 2,20-Azoisobutyronitrile (AIBN) was recrystallized from 95%
ethanol. N,N0-Dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyr-
idine (DMAP), 2-chloroethanol, ethyl acetoacetate, acetic acid (AcOH),
tetrabutylammonium bromide (TBAB), tetrabutylammonium fluoride
(TBAF), lithium chloride (LiCl, anhydrous), 3,4-dihydro-2H-pyran
(DHP), tert-butyldimethylsilyl chloride (TBSCl), 4- methylbenzenesul-
fonic acid (TsOH), diethyl malonate, ethyl acetate (EtOAc), and all
other reagents were purchased from Sinopharm Chemical Reagent Co.
Ltd. and used as received. Water was deionized with a Milli-Q SP reagent
water system (Millipore) to a specific resistivity of 18.4 MΩ cm.
2-Succinyloxyethyl methacrylate (SEMA) was synthesized via the
esterification reaction of 2-hydroxyethyl methacrylate (HEMA) with
succinic anhydride according to literature procedures.60 S-1-Propyl-
S0-(α,α0-dimethyl-α00-acetic acid)- trithiocarbonate (PDMAT) and PEO113
-
based macro-RAFT agent (PEO-CTA) were prepared via the esterification
reaction of PEO113ꢀOH with PDMAT in the presence of DCC and
DMAP.51
Sample Preparation. Synthetic schemes employed for the pre-
paration of fluorescence “turn-on” small molecule precursor, diethyl
2-(2-((tert-butyldimethylsilyl)oxy)-4-(2- hydroxylethoxy)benzylidene)-
malonate (SiCouꢀOH, 7) and probe-functionalized double hydrophilic
diblock copolymer, PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA (8),
are shown in Schemes 2 and 3, respectively.
’ EXPERIMENTAL SECTION
Synthesis of 4-(2-Hydroxyethoxy)-2-Methoxybenzaldehyde (2). TBAB
(0.64 g, 2 mmol), 4-hydroxy-2-methoxybenzaldehyde (1, 22.82 g, 0.15
mol), and NaOH (12.0 g, 0.30 mol) were dissolved in 300 mL absolute
EtOH under nitrogen atmosphere. The reaction mixture was heated to
reflux, and 2-chloroethanol (24.15 g, 0.30 mol) was added slowly via a
dropping funnel within ∼1 h. After stirring overnight under reflux, the
reaction mixture was allowed to cool to room temperature and filtered to
remove precipitated salts. After evaporating all the solvents, the residues
were dissolved in CH2Cl2, successively washed with 10% aqueous
NaOH and water, dried over anhydrous MgSO4, filtered, and then
concentrated on a rotary evaporator. The crude product was subjected to
Materials. Poly(ethylene oxide) monomethyl ether (PEO113ꢀOH,
Mn = 5.0 kDa, Mw/Mn = 1.06, mean degree of polymerization, DP, is
113) was purchased from Aldrich and used as received. Oligo(ethylene
glycol) methyl ether methacrylate (OEGMA, Mn = 475, DP is 8ꢀ9;
Aldrich) and di(ethylene glycol) methyl ether methacrylate (MEO2MA,
95%, Aldrich) were passed through an alumina column to remove the
inhibitor. All purified monomers were stored at ꢀ20 ꢀC prior to use. 1,4-
Dioxane, ethyl ether, and tetrahydrofuran (THF) were dried by refluxing
over sodium/benzophenone and distilled prior to use. Dichloromethane
(CH2Cl2), acetonitrile (MeCN), and pyridine were dried over CaH2
8782
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
Scheme 3. Reaction Schemes Employed for the Synthesis of Double Hydrophilic Diblock Copolymers Bearing Fꢀ-Reactive Moieties
(SiCouMA) within the Thermoresponsive Block, PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA (8), via RAFT Polymerizationa
a In the presence of Fꢀ ions, deprotection reaction followed by cyclization of SiCouMA moieties leads to the formation of highly fluorescent by
employing PEO-b-P(MEO2MA-co-OEGMA-co-CouMA) (9) bearing coumarin residues in the thermoresponsive block.
further purification by silica gel column chromatography using CH2Cl2 as
the eluent, affording 4-(2-hydroxyethoxy)-2-methoxybenzaldehyde (2)
as a yellowish solid (22.7 g, 77% yield). 1H NMR (CDCl3, δ, ppm, TMS):
10.27 (s, 1H, ꢀCHO), 7.79, 6.54, and 6.48 (m, 3H, aromatic protons),
4.16 (t, 2H, ꢀOCHH2CH2OH), 4.00 (t, 2H, ꢀOCHH2CH2OH), 3.89
(s, 3H, ꢀOCHH3), 1.74 (br, 1H, ꢀOCHH2CH2OH).
(t, 2H, ꢀOCHH2CH2OH), 3.99 (t, 2H, ꢀOCHH2CH2OH), 2.00 (br,
1H, ꢀOCHH2CH2OH). 13C NMR (CDCl3, δ, ppm, TMS; Figure S1,
Supporting Information): 194.63, 164.23, 155, 37, 135.31, 108.80, 101.42,
69.54, 64.64, 61.22.
Synthesis of 2-Hydroxy-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)-
benzaldehyde (4). To a stirred solution of 3 (3.64 g, 0.020 mol) and
DHP (2.52 g, 0.030 mol) in CH2Cl2 (60 mL) at 25 ꢀC, TsOH (20 mg,
0.12 mmol) was added and the reaction mixture was stirred for 2 h. The
reaction mixture was washed with saturated aqueous NaCl solution
(60 mL) for three times and dried with Na2SO4, and concentrated in
vacuo. The residues obtained were subjected to flash column chroma-
tography (silica gel, CH2Cl2) to afford 2-hydroxy-4-(2-((tetrahydro-2H-
pyran-2-yl)oxy)ethoxy)benzaldehyde (4) as a yellow liquid (4.4 g, 83%
Synthesis of 4-(2-Hydroxyethoxy)salicylaldehyde (3). A 500 mL
round-bottom flask was charged with 2 (19.6 g, 0.10 mol), anhydrous
LiCl (12.7 g, 0.30 mol), and DMF (200 mL). The mixture were heated
to reflux under nitrogen atmosphere for 48 h, and then the reaction
mixture was allowed to cool to room temperature, followed by the
addition of 600 mL 10% aqueous NaOH. The solution pH was adjusted
to ∼2. This was followed by extraction with EtOAc (100 mL ꢁ 3). The
combined organic layers were washed with saturated brine, dried over
anhydrous MgSO4, filtered, and then concentrated on a rotary evapora-
tor. The crude product was purified by silica gel column chromatography
using CH2Cl2 as eluent, affording 4-(2-hydroxyethoxy)salicylaldehyde
1
yield). H NMR (CDCl3, δ, ppm, TMS; Figure S2, Supporting Infor-
mation): 11.45 (s, 1H, ArꢀOH), 9.72 (s, 1H, ꢀCHO), 7.43, 6.56, and
6.43 (m, 3H, aromatic protons), 4.21 (m, 2H, ꢀOCHH2CH2OTHP),
3.85 (m, 2H, ꢀOCHH2CH2OTHP), 4.70, 3.53, 1.83, 1.74, 1.58 (m, 9H,
ꢀOTHP). 13C NMR (CDCl3, δ, ppm, TMS; Figure S2, Supporting
Information): 194.16, 166.30, 164.58, 135.00, 115.07, 108.85, 101.45,
99.20, 67.82, 65.56, 62.14, 30.30, 25.16, 19.48.
1
(3) as a white crystal (9.0 g, 49% yield). H NMR (CDCl3, δ, ppm,
TMS; Figure S1, Supporting Information): 11.45 (s, 1H, ArꢀOH), 9.72
(s, 1H, ꢀCHO), 7.43, 6.56, and 6.43 (m, 3H, aromatic protons), 4.13
8783
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
Synthesis of 2-((tert-Butyldimethylsilyl)oxy)-4-(2-((tetrahydro-2H-
pyran-2-yl)oxy)ethoxy)benzaldehyde (5). A 250 mL round-bottom
flask was charged with 4 (2.66 g, 0.01 mol), imidazole (0.817 g, 0.012 mol),
and anhydrous CH2Cl2 (100 mL), and then TBSCl (1.81 g, 0.012 mol)
dissolved in 20 mL of anhydrous CH2Cl2 was slowly added dropwise
over ∼1 h. The reaction mixture was stirred at room temperature for
24 h. After that, the mixture was washed with saturated aqueous NaCl
solution (100 mL) for three times, dried with anhydrous Na2SO4, and
then concentrated in vacuo. The residues obtained were subjected to
flash column chromatography (silica gel, CH2Cl2) to give 2-((tert-
butyldimethylsilyl)oxy)-4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethoxy)-
benzaldehyde (5) as a yellow liquid (3.5 g, 92% yield). 1H NMR
(CDCl3, δ, ppm, TMS; Figure S3, Supporting Information): 10.28 (s,
1H, ꢀCHO), 7.76, 6.60, 6.35 (m, 3H, aromatic protons), 4.17 (m, 2H,
ꢀOCHH2CH2OTHP), 3.84 (m, 2H, ꢀOCHH2CH2OTHP), 4.68,
3.52, 1.81, 1.73, 1.56 (m, 9H, -OTHP), 1.00 (s, 6H, ꢀOSi(CH3)2C-
(CH3)3), 0.27 (s, 9H, ꢀOSi(CH3)2C(CH3)3). 13C NMR (CDCl3, δ,
ppm, TMS; Figure S3, Supporting Information): 188.39, 165.26, 160.58,
129.92, 121.20, 108.54, 105.60, 99.27, 67.97, 65.68, 62.19, 30.34, 25.66,
25.11, 19.33, 18.13, ꢀ4.45.
Synthesis of 2-((tert-Butyldimethylsilyl)oxy)-4-(2-hydroxyethoxy)-
benzaldehyde (6). A 100 mL round-bottom flask was charged with 5
(2.28 g, 0.006 mol), MgBr2 (3.32 g, 0.018 mol), and anhydrous ethyl
ether (50 mL), and the reaction mixture was stirred at room temperature
for 2 h and then washed with saturated aqueous NaCl solution (50 mL)
three times. After drying with anhydrous Na2SO4 and removing all the
solvents under reduced pressure, the residues obtained were subjected to
flash column chromatography (silica gel, CH2Cl2) to give 2-((tert-
butyldimethylsilyl)oxy)-4-(2-hydroxyethoxy)benzaldehyde (6) as a yel-
low liquid (1.5 g, 84% yield). 1H NMR (CDCl3, δ, ppm, TMS; Figure
S4, Supporting Information): 10.30 (s, 1H, ꢀCHO), 7.80, 6.59, 6.38
(m, 3H, aromatic protons), 4.11 (t, 2H, ꢀOCHH2CH2OH), 3.99 (t,
2H, ꢀOCHH2CH2OH), 1.96 (br, 1H, ꢀOCHH2CH2OH), 1.02, (s,
Figure 1. 1H NMR and 13C NMR spectra recorded in CDCl3 for an Fꢀ-
reactive small molecule precursor (SiCouꢀOH, 7).
MEO2MA (2.71 g, 14.4 mmol), OEGMA (0.285 g, 0.60 mmol), SEMA
(0.138 g, 0.60 mmol), PEO113ꢀCTA (0.783 g, 0.15 mmol), AIBN
(0.006 g, 0.04 mol), and 1,4-dioxane (6 mL). The reaction tube was
carefully degassed by three freezeꢀpumpꢀthaw cycles and then sealed
under vacuum. After thermostating at 60 ꢀC in an oil bath and stirring for
10 h, the reaction tube was quenched into liquid nitrogen, opened, and
diluted with 1,4-dioxane. The mixture was then precipitated into an
excess of diethyl ether. The above dissolutionꢀprecipitation cycle was
repeated for three times. PEO-b-P(MEO2MA-co-OEGMA-co-SEMA)
was obtained as a white solid (2.30 g, 58% yield). GPC analysis revealed
an Mn of 23.3 kDa and an Mw/Mn of 1.15 (Figure S5, Supporting
Information). The actual DP of P(MEO2MA-co-OEGMA-co-SEMA)
block (93), together with the molar contents of three comonomers were
6H, -OSi(CH3)2C(CH3)3), 0.29, (s, 9H, ꢀOSi(CH3)2C(CH3)3). 13
C
NMR (CDCl3, δ, ppm, TMS; Figure S4, Supporting Information):
188.54, 164.49, 160.88, 130.36, 121.90, 108.11, 105.74, 69.71, 61.34,
25.31, 18.18, ꢀ4.07.
Synthesis of Diethyl 2-(2-((tert-Butyldimethylsilyl)oxy)-4-(2-Hydro-
xyethoxy)benzylidene)malonate (7). A 100 mL round-bottom flask was
charged with 20 mL of anhydrous THF. After cooling to 0 ꢀC in an
iceꢀwater bath, TiCl4 (1.55 g, 0.008 mol) in 2 mL anhydrous CCl4 was
then added slowly at 0 ꢀC. 6 (1.21 g, 0.004 mol) and diethyl malonate
(0.65 g, 0.004 mol) in 5 mL of anhydrous THF was then added dropwise
over ∼0.5 h. Subsequently pyridine (1.32 g, 0.016 mol) in 2 mL of THF
was added dropwise at 0 ꢀC. The reaction mixture was stirred at 0 ꢀC for
another 0.5 h and then quenched with saturated aqueous NaCl solution
and washed with saturated aqueous NaCl solution (50 mL) for three
times. The combined organic phases were dried over anhydrous Na2SO4
and concentrated in vacuo. The residues obtained were subjected to
flash column chromatography (silica gel, CH2Cl2) to afford 2-(2-((tert-
butyldimethylsilyl)oxy)-4-(2-hydroxyethoxy)benzylidene)malonate (7) as
1
determined by H NMR analysis. The final product was denoted as
PEO113-b-P(MEO2MA0.924-co-OEGMA0.039-co-SEMA0.037)93.
Synthesis of PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA) (8). The
reactive probe- functionalized double hydrophilic diblock copolymer 8
was prepared by the postmodification of PEO113-b-P(MEO2MA0.924-co-
OEGMA0.039-co-SEMA0.037 93
DMAP. In a typical procedure, PEO113-b-P(MEO2MA0.924-co-OEG-
MA0.039-co- SEMA0.037 93 (1.20 g, 0.17 mmol carboxyl moieties) was
)
with 7 in the presence of DCC and
)
1
a yellow liquid (1.2 g, 69% yield). H NMR (CDCl3, δ, ppm, TMS;
dissolved in anhydrous toluene (10 mL), and then azeotropic distillation
was carried out at 50 ꢀC under reduced pressure to remove most of the
solvent. 7 (0.011 g, 0.025 mmol) and dry CH2Cl2 (40 mL) were then
added. After cooling to 0 ꢀC in an iceꢀwater bath, a mixture containing
DCC (0.51 g, 0.25 mmol), DMAP (0.003 g, 0.025 mmol), and dry
CH2Cl2 (10 mL) was added dropwise over ∼1 h. The reaction mixture
was stirred at room temperature for 24 h. After removing insoluble salts
by filtration, the filtrates were concentrated on a rotary evaporator and
then precipitated into an excess of cold diethyl ether. The above
dissolutionꢀprecipitation cycle was repeated for three times. After
drying in a vacuum oven overnight at room temperature, PEO-b-
P(MEO2MA-co-OEGMA-co- SiCouMA) (8) was obtained as a yellow
solid (1.02 g, yield: 86%). The actual molar content of SiCouMA
Figure 1): 7.97, (s, 1H, ArꢀCHC(COOCH2CH3)2), 7.70, 6.60, 6.37,
(m, 3H, aromatic protons), 4.29, (m, 4H, ArꢀCHC(COOCH2CH3)2),
4.11, (t, 2H, ꢀOCHH2CH2OH), 3.99, (t, 2H, ꢀOCHH2CH2OH), 1.96,
(br, 1H, ꢀOCHH2CH2OH), 1.30, (m, 6H, ArꢀCHC(COOCH2CH3)2),
1.00, (s, 9H, -OTBS), 0.29, (s, 9H, -OTBS). 13C NMR (CDCl3, δ, ppm,
TMS; Figure 1): 167.30, 164.60, 161.80, 156.70, 137.42, 129.87, 118.14,
107.82, 106.16, 69.33, 61.37, 25.71, 18.26, 14.21, ꢀ4.31.
Synthesis of PEO-b-P(MEO2MA-co-OEGMA-co-SEMA) Thermore-
sponsive Double Hydrophilic Diblock Copolymer. Typical procedures
employed for the RAFT synthesis of thermoresponsive diblock copoly-
mer, PEO-b-P(MEO2MA-co-OEGMA-co-SEMA), are as follows. Into
a reaction tube equipped with a magnetic stirring bar were charged
8784
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
residues in 8 was determined by UVꢀvis absorption spectroscopy in
EtOH by using 7 as the calibration standard.
diblock precursor was denoted as PEO113-b-P(MEO2MA0.924-
co-OEGMA0.039-co-SEMA0.037)93. On average, there exist ∼3.44
Characterization. All nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker AV300 NMR spectrometer (resonance
carboxyl functionalities per diblock copolymer chain.
On the basis of the overall design as depicted in Schemes 1ꢀ3,
the key point in the current work is the efficient synthesis of
Fꢀ ion-reactive fluorescence “turn-on” small molecule building
block, 7, which possesses one hydroxyethyl functionality for the
covalent attachment with carboxyl moieties within PEO113-b-
P(MEO2MA0.924-co-OEGMA0.039-co-SEMA0.037)93. Most im-
portantly, probe 7 contains tert-butyldimethylsilyl protected
diethyl 2-(benzylidene)- malonate motif. As discussed in the
introduction part, the chemical design of 7 was inspired by the
work of Swager et al.58 In the presence of Fꢀ ions, 7 is subjected
to desilylation reaction, accompanied by the release of substi-
tuted phenol moieties; this step was spontaneously followed by
the cyclization reaction to form highly fluorescent coumarin
derivative (see Schemes 1 and 3 for details). Using 4-hydroxy-2-
methoxybenzaldehyde (1) as the starting material,7 was synthe-
sized via six steps with an overall yield of 16.7%. 1H NMR and 13C
NMR spectra of key intermediates, 3, 4, 5, and 6, are shown in
Figures S1, S2, S3, and S4, Supporting Information, respectively,
1
frequency of 300 MHz for H and 75 MHz for 13C) operated in the
Fourier transform mode. CDCl3 was used as the solvent. Molecular
weights and molecular weight distributions were determined by gel
permeation chromatography (GPC) equipped with a Waters 1515 pump
and a Waters 2414 differential refractive index detector (set at 30 ꢀC). It
used a series of two linear Styragel columns (HR2 and HR4) at an oven
temperature of 45 ꢀC. The eluent was THF at a flow rate of 1.0 mL/min.
A series of low-polydispersity polystyrene standards were employed for
calibration. All UVꢀvis spectra were acquired on a Unico UV/vis
2802PCS spectrophotometer. Concerning temperature-dependent tur-
bidimetry, the optical transmittance of aqueous solutions at a wavelength
of 600 nm was acquired on a Unico UV/vis 2802PCS spectrophot-
ometer. A thermostatically controlled couvette was employed and
the heating rate was 0.2 ꢀC minꢀ1. The LCST was defined as the
temperature corresponding to ∼1% decrease of optical transmittance.
A commercial spectrometer (ALV/DLS/SLS-5022F) equipped with a
multitau digital time correlator (ALV5000) and a cylindrical 22 mW
UNIPHASE HeꢀNe laser (λ0 = 632 nm) as the light source was
employed for dynamic laser light scattering (LLS) measurements.
Scattered light was collected at a fixed angle of 90ꢀ for duration of
∼10 min. Distribution averages and particle size distributions were
computed using cumulants analysis and CONTIN routines. All data
were averaged over three measurements. Fluorescence spectra were
recorded using a RF-5301/PC (Shimadzu) spectrofluorometer. The
temperature of the water-jacketed cell holder was controlled by a
programmable circulation bath. The slit widths were set at 5 nm for
excitation and 5 nm for emission.
1
together with the peak assignments. H NMR and 13C NMR
spectra of the target compound, 7, are shown in Figure 1. All
resonance signals can be well-assigned and peak integral ratios
confirmed the successful synthesis of 7.
With the small molecule Fꢀ ion-reactive probe 7 in hand, we
then covalently attached 7 onto the thermoresponsive block of
PEO113-b-P(MEO2MA0.924-co-OEGMA0.039-co- SEMA0.037 93
)
via esterification reaction of 7 with carboxyl moieties within
the diblock copolymer precursor. To ensure complete consump-
tion of 7 in the reaction mixture, the [hydroxyl]/[carboxyl] feed
molar ratio was kept to be quite low (∼1/6.8). Note that it is also
quite advantageous to keep the labeling content of 7 within the
thermoresponsive block at a relatively low level. If the labeling
density on the diblock chain is too high, undesirable partial
fluorescence quenching among Fꢀ ion-generated coumarin
species might occur;63 moreover, a high labeling content of 7
this will also incur considerable changes in the critical micelliza-
tion temperature (CMT) of the thermoresponsive diblock
copolymer. 1H NMR spectrum of PEO-b-P(MEO2MA-co-OEG-
MA-co-SiCouMA) (8) in CDCl3 is shown in Figure S6, Support-
ing Information. The presence of resonance signals characteristic
of SiCouMA residues can be clearly discerned. The labeling
density of SiCouMA on the final product, PEO-b-P(MEO2MA-
co-OEGMA-co- SiCouMA) (8), was calculated based on UVꢀvis
absorption by using 7 as the calibration standard. For polymer 8
in ethanol at a concentration of 0.5 g/L, the SiCouMA concen-
tration is determined to be ∼5.3 μM.
’ RESULTS AND DISCUSSION
Synthesis and Thermo-Induced Aggregation of PEO-b-
P(MEO2MA-co-OEGMA-co-SiCouMA) (8). PMEO2MA homo-
polymers and its random copolymers with OEGMA (DP = 8ꢀ9)
are well-known to be thermoresponsive in aqueous solution and
possess lower critical solution temperatures (LCST), which are
quite similar to that exhibited by poly(N-isopropylacrylamide)
(PNIPAM) in aqueous solution.61 In the current case, well-
defined double hydrophilic diblock copolymer precursor bearing
carboxyl moieties in the thermoresponsive block, PEO-b-P-
(MEO2MA-co-OEGMA-co-SEMA), was synthesized at first via
the RAFT copolymerization of MEO2MA, OEGMA, and SEMA
comonomers using PEO113ꢀCTA as the macro-RAFT agent.
The controlled RAFT polymerization of methacrylates mono-
mers such as MEO2MA and OEGMA has been well-documented
in literature reports.61,62 Compared to that of PEO113ꢀCTA
macro-RAFT agent, the GPC elution trace of PEO-b-P-
(MEO2MA-co-OEGMA-co-SEMA) precursor clearly shifted to
the higher molecular weight (MW) side. Moreover, the elution
peak is relatively sharp and symmetric, exhibiting no tailing at the
lower molecular weight side, suggesting the high initiating
efficiency of PEO113-based macro-RAFT agent. GPC analysis
of the carboxyl-functionalized diblock precursor revealed an Mn
of 23.3 kDa and an Mw/Mn of 1.15 (Figure S5, Supporting
Information). The actual DP of P(MEO2MA-co-OEGMA-co-
The thermoresponsive micellization behavior of PEO-b-P-
(MEO2MA-co-OEGMA-co- SiCouMA) (8) in aqueous solution
was then investigated by temperature-dependent optical trans-
mittance and dynamic LLS measurements. Note that SiCouMA
moieties in 8 exhibit slight reactivity even in neutral aqueous
media, which will be discussed in detail in subsequent sections.
To avoid possible interference from this, all UVꢀvis and
dynamic LLS measurements were immediately carried out when
freshly prepared samples are ready. Figure 2a shows the tem-
perature-dependent optical transmittance of 8 in aqueous solu-
tion (1.0 g/L). It was found that the optical transmittance
remains almost constant at temperatures below 32 ꢀC.61 Above
33 ꢀC, the optical transmittance exhibits abrupt decrease, accom-
panied by the appearance of a bluish tinge, which is characteristic
1
SEMA) block was determined to be 93 based on H NMR
analysis in CDCl3. 1H NMR analysis also revealed that within the
thermoresponsive P(MEO2MA-co-OEGMA-co-SEMA) block,
the molar fraction of three comonomers, MEO2MA, OEGMA,
and SEMA are 0.924, 0.039, and 0.037, respectively. Thus, the
8785
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
Figure 2. (a) Temperature dependence of optical transmittance at a
wavelength of 600 nm recorded for 1.0 g/L aqueous solution of PEO-b-
P(MEO2MA-co-OEGMA-co-SiCouMA) (8) bearing Fꢀ-reactive moi-
eties within the thermoresponsive block. (b) Hydrodynamic radius
distributions, f(Rh), recorded for 0.1 g/L aqueous solution of 8 at 20
and 40 ꢀC.
Figure 3. (a) Time-dependent changes in fluorescence emission in-
tensities at 402 nm recorded for THF solution of 8 (0.5 g/L, 20 ꢀC)
upon addition of 100.0 equiv of TBAF and NaF, respectively. (b) Time-
dependent evolution of fluorescence emission spectra recorded for the
aqueous solution of PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA) (8,
0.5 g/L, 5.3 μM SiCouMA moieties; 20 mM phosphate buffer; 20 ꢀC)
upon addition of 10.0 equiv. NaF (relative to SiCouMA moieties). The
inset in (b) shows optical images recorded under UV 365 nm irradiation
for aqueous solutions of 8 (0.5 g/L) (I) in the absence and (II) in the
presence of NaF (10.0 equiv; 420 min after addition), respectively.
of colloidal dispersions. This suggests the occurrence of thermo-
induced micellization due the thermoresponsiveness of P(MEO2MA-
co-OEGMA-co- SiCouMA) block. Thus, above the CMT of
33 ꢀC, 8 will self-assemble into micellar nanoparticles possessing
hydrophobic P(MEO2MA-co-OEGMA-co-SiCouMA) cores sta-
bilized by well-solvated PEO coronas (Scheme 1). The thermo-
induced aggregation of 8 was further investigated by dynamic
LLS. Figure 2b shows hydrodynamic radius distribution, f(Rh),
of 8 in aqueous solution (0.1 g/L) at 20 and 40 ꢀC, respectively.
At 20 ꢀC, the intensity-average hydrodynamic radius, ÆRhæ, is
∼9.8 nm, suggesting that diblock copolymers exist as unimole-
cularly dissolved chains. Upon heating to 40 ꢀC, the Rh distribu-
tion clearly shifts to higher values, affording an ÆRhæ of ∼31.2 nm
and a polydispersity, μ2/Γ2, of 0.08. This indicates that relatively
monodisperse nanosized micellar nanoparticles were formed
from PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA) (8) at 40 ꢀC,
which is well above the CMT of diblock copolymers.
intensity remains at constantly low background values, suggest-
ing that SiCouMA residues in 8 is quite stable and no reaction
have occurred. It was found that for 0.5 g/L THF solution of 8
(5.3 μM SiCouMA moieties) in the presence of 100.0 equiv of
TBAF (final concentration 10.0 ppm), the emission intensity at
402 nm exhibit monotonic increase with the incubation time,
suggesting the generation of coumarin moieties induced by
fluoride ions. We can also observe that even at 400 min after
the addition of TBAF, the emission intensity is still gradually
increasing, suggesting that in THF, the reaction is quite slow.
These preliminary results strongly suggest that 8 can act as a
reactive fluorescence “turn-on” probe for TBAF.
Considering the poor bioavailability of organic TBAF salts and
that most fluoride ions exist in the form sodium or potassium
salts, next, we attempted to utilize the THF solution of 8 to detect
NaF in a fluorometric manner. It is not strange that the addition
of 100.0 equiv. NaF to 8 in THF essentially does not lead to any
fluorescence emission enhancement, presumably due to the poor
solubility of NaF in THF (Figure 3a). The addition of KF results
in the same phenomenon as that of NaF. Since the detection,
sensing, and imaging of Fꢀ ions in drinking water and other bio-
logical samples (e.g., cells, tissues, and organs) are solely dealing
with aqueous media, in the next section, we put emphasis on the
fluorometric sensing of fluoride ions in purely aqueous media.
The general design principle is shown in Scheme 1. The
covalent embedment of water-insoluble SiCouMA moieties into
the thermoresponsive block of PEO-b-P(MEO2MA- co-OEG-
MA-co-SiCouMA) (8) provides the advantage of much more
PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA) Diblock Co-
polymer (8) Unimers and Micelles as Reactive Fluorescence
“Turn-On” Probes for Fꢀ Ions in Purely Aqueous Media.
Previously, Swager et al.58 covalently attached tert-butyldimethyl-
silyl protected diethyl 2-(benzylidene)malonate moiety (as con-
tained in 7, see Scheme 3) onto polythiophene conjugated
polymers to achieve amplified fluorometric Fꢀ ion detection.
In THF solution, the addition TBAF leads to the deprotection of
trialkylsilyl moieties and the subsequent generation of highly
fluorescent coumarin side groups via cyclization. Fꢀ ion-induced
interconnection of the new transduction pathway to the fluor-
escent conjugated backbone resulted in ∼100-fold enhancement
of detection sensitivity. In the current work, we first investigated
the Fꢀ ion sensing capability of reactive PEO-b-P(MEO2MA-co-
OEGMA-co-SiCouMA) (8) in organic solvents, and the results
are shown in Figure 3a. In the absence of TBAF, the emission
8786
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
due to competitive hydrolysis reactions by water molecules and
by fluoride ions.40ꢀ42,44,46 In the current design, the fluorescence
“turn-on” involves two steps, namely, deprotection of tert-
butyldimethylsilyl and cyclization reaction into the highly fluores-
cent coumarin moieties. As reported by Swager et al.,58 in THF,
the second step is the rate-determining one at Fꢀ concentrations
less than ∼3.0 equiv relative to that of SiCouMA residues. We
suppose that this characteristic feature might be advantageous for
the use of 8 as aqueous media-based fluorometric “turn-on”
sensor for fluoride ions as the background emission intensity
solely caused by spontaneous hydrolysis by water molecules can
be retarded to some extent.
In the absence of NaF, the time evolution of emission intensity
at 402 nm for 8 in aqueous media (pH 7.4) was then monitored
at 20 and 40 ꢀC for diblock unimers and micelles, respectively. As
shown in parts a and b of Figure 4, emission intensities of the
aqueous solution of 8 indeed exhibit slight increase with increas-
ing incubation time duration. Diblock micelles at 40 ꢀC exhibit
more prominent increase with time compared to that 20 ꢀC.
However, we can clearly tell from Figure 4, parts a and b, that for
diblock unimers and micelles at 20 and 40 ꢀC, the presence of
0.2 and 1.0 ppm of NaF can both accelerate the fluorescence
“turn-on” process, as evidenced by prominently elevated slopes
of emission intensity-incubation time curves.
On the basis of the above results, we conclude that although
the fluorescence intensity of the aqueous solution of 8 could be
enhanced with increasing incubation time in the presence of
NaF, extended duration of incubation will surely incur the
increase of undesirable contribution by the water hydrolysis
pathway. To effectively eliminate the premature fluorescence
“turn-on” by the background aqueous media and to obtain
reliable, consistent, and comparable analytical data, and to
achieve fast fluoride ion detection, in subsequent sections, the
incubation time was fixed to be 20 min after the addition of
Fꢀ ions.43ꢀ46 During this time period, the background signal
intensity incurred by water molecules can be kept at a
minimum level.
For the aqueous solution of PEO-b-P(MEO2MA-co-OEGMA-
co-SiCouMA) (8) at a concentration of 0.5 g/L and 20 ꢀC, the
incubation time was fixed at 20 min after the addition of 0ꢀ1600
equiv of NaF (relative to SiCouMA residues), and the NaF
concentration dependence of emission spectra are shown in
Figure 5a. We can see that in the range of 0ꢀ1600 equiv of NaF,
the emission intensity at 402 nm exhibits ∼88-fold increase.
Most importantly, in the low NaF concentration range, there
exists excellent linear relationship (R = 0.9989) between the
emission intensity and NaF concentrations in the range of
0ꢀ15 ppm for diblock unimers at 20 ꢀC with the coefficient
(inset in Figure 5b).
At 40 ꢀC, the diblock copolymer 8 spontaneously forms
micellar nanoparticles. In the same NaF concentration range
(0ꢀ1600 equiv), theemissionintensityat402nmexhibits∼30-fold
enhancement compared to the blank sample (Figure 6). More-
over, the same linear relationship as observed for that of diblock
unimers at 20 ꢀC can be again obtained in the NaF concentration
range of 0ꢀ8 ppm for diblock micelles at 40 ꢀC (inset in
Figure 6b). The ∼30-fold emission enhancement for diblock
micelles at 40 ꢀC is less potent than that at 20 ꢀC for diblock
unimers in the same range of NaF concentration (0ꢀ1600 equiv),
this might be partially ascribed to the increase of background
signal intensity incurred by the water hydrolysis pathway, as
evidenced by the much faster increase of emission intensity with
Figure 4. Time-dependent changes in fluorescence emission intensities
at 402 nm recorded for the aqueous solution of 8 (0.5 g/L, 5.3 μM
SiCouMA moieties; 20 mM phosphate buffer) at (a) 20 ꢀC and (b) 40 ꢀC
upon addition of 2.0 and 10.0 equiv of NaF.
improved water solubility. Moreover, 8 can exist as unimers
below the CMT and self-assemble into coreꢀshell type micellar
nanoparticle at elevated temperature due to the thermorespon-
siveness of SiCouMA-labeled block. The formation of micelles
can effectively encapsulate fluoride ion-reactive SiCouMA and
Fꢀ-generated highly fluorescent CouMA within the hydrophobic
cores, thus, multiple advantages such as the modulation of detec-
tion sensitivity, linear detection range, and possible integration of
temperature detection capability are envisaged to be achieved.
We then monitored the time-evolution of fluorescence emis-
sion spectra recorded for the aqueous solution of 8 (0.5 g/L,
pH 7.4) at 20 ꢀC upon addition of 10.0 equiv of NaF (relative to
SiCouMA moieties) and the results are shown in Figure 3b.
Apparently, we can discern the prominent enhancement of
emission at around 402 nm with the increase of incubation time.
From the inset in Figure 3b, the presence of 10 equiv. (1.0 ppm)
NaF clearly leads to the fluorometric transition from nonemissive
to intense blue emission, which can be easily checked by the
naked eye. Thus, at 20 ꢀC, diblock unimers provides a facile
detection approach for NaF. From Figure 4a, it can be quantified
that the addition of 1.0 ppm (10.0 equiv) NaF leads to ∼69.2-fold
increase in emission intensity in the time range of 0ꢀ420 min.
In the presence of 0.2 ppm of NaF, ∼28.8-fold increase in the
fluorescence intensity can be observed in the same time period.
Moreover, the emission intensityꢀincubation time plot in the
presence of 1.0 ppm of NaF possesses appreciably larger slope that
in the presence of 0.2 ppm of NaF. This strongly suggests that
diblock unimers of 8 can serve as reactive fluorescence “turn-on”
probe for NaF in purely aqueous media.
Despite of the above positive results, we are quite conscious of
the reactiveness of SiCouMA moieties in 8 toward water molecules
even under neutral aqueous media (pH 7.4 buffer). The water
reactivity of trialkylsilyl protecting groups has actually led to quite
high background signal intensity for those trialkylsilyl-caged dyes
8787
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
Figure 6. (a) Fluorescence emission spectra and (b) changes in relative
fluorescence intensities at 402 nm recorded for the aqueous solution of
PEO-b-P(MEO2MA-co- OEGMA-co-SiCouMA) (8, 0.5 g/L, 5.3 μM
SiCouMA moieties; 20 mM phosphate buffer; 40 ꢀC) at 20 min after the
addition of 0ꢀ1600 equiv of NaF. The inset (c) shows the enlarged
region of part b in the range of 0ꢀ80 equiv (0ꢀ8 ppm) of NaF. All data
points in parts b and c are averaged values from at least three parallel
measurements.
Figure 5. (a) Fluorescence emission spectra and (b) changes in relative
fluorescence intensities at 402 nm recorded for the aqueous solution of
PEO-b-P(MEO2MA-co- OEGMA-co-SiCouMA) (8, 0.5 g/L, 5.3 μM
SiCouMA moieties; 20 mM phosphate buffer; 20 ꢀC) at 20 min after the
addition of 0ꢀ1600 equiv. NaF. The inset (c) shows the enlarged region
of part b in the range of 0ꢀ150 equiv (0ꢀ15 ppm) of NaF. All data
points in parts b and c are averaged values from at least three parallel
measurements.
incubation time for blank samples at 40 ꢀC compared to that at
20 ꢀC (Figure 4, parts a and b).
of emission intensity with increasing temperature (Figure S8a,
Supporting Information). Most importantly, an almost linear
relationship (R = 0.997) between the relative fluorescence
intensity (I/I0) and temperature was obtained in the range of
20ꢀ60 ꢀC (Figure S8b, Supporting Information). The same pheno-
menon has been previously observed for coumarin-functionalized
poly(vinyl alcohol).64 On the basis of these results, PEO-b-
P(MEO2MA-co-OEGMA-co-SiCouMA) (8) is also endowed with
the fluorometric temperature sensing function, in addition to
serving as a highly sensitive chemical reaction-based fluorescence
“turn-on” probe for fluoride ions in purely aqueous media.
Finally, the detection selectivity of 8 for fluoride ions was
A direct comparison of results shown in Figure 6b with that in
Figure 5b reveals that for diblock micelles at 40 ꢀC, the addition
of ∼400 equiv of NaF can result in the complete consumption of
SiCouMA moieties in 8 within just 20 min; whereas for diblock
unimers at 20 ꢀC, ∼1200 equiv. NaF need to be added to achieve
comparable conversion of reactive SiCouMA species. This im-
plies that NaF detection using diblock micelles at 40 ꢀC can
achieve considerably faster response time. If we arbitrarily define
the detection limit as the NaF concentration at which a 10%
fluorescence enhancement can be measured by employing 0.5 g/L
aqueous solution of 8, the detection limit toward Fꢀ ions was
determined to be ∼0.065 ppm at 20 ꢀC for diblock unimers; for
diblock micelles at 40 ꢀC, the detection limit is slightly improved
to ∼0.05 ppm (Figure 7, parts a and b). Considering the practical
application of Fꢀ ion sensing in drinking water or relevant
biological media, fluorescence emission spectra and changes in
relative intensities at 402 nm were also recorded for the micellar
solution of 8 at 37 ꢀC (Figure S7, Supporting Information). It
was found that the fluorometric Fꢀ ion-sensing performance is
quite comparable to that occurred at 40 ꢀC (Figure 6), with the
detection limit being ∼0.053 ppm.
examined. At a polymer concentration of 0.5 g/L, 100 equiv of
2‑
sodium salts of Clꢀ, Brꢀ, Iꢀ, CO32‑, NO3ꢀ, HSO4ꢀ, HPO4
,
H2PO4ꢀ, AcOꢀ, and Fꢀ were added, respectively. It was observed
that 20 min after addition, only Fꢀ ions exhibit prominent fluor-
escence emission enhancement, and no other ions induce dis-
cernible changes in emission intensities (Figure 8). This clearly
verified the high selectivity of diblock copolymer 8 as a chemical
reaction-based fluorescence “turn-on” probe for fluoride ions in
aqueous media.
On the basis of the above results, we expect that the fluoro-
metric sensing of Fꢀ ions by PEO-b-P(MEO2MA-co-OEGMA-
co-SiCouMA) unimers and micelles in purely aqueous media
should be applicable in practical circumstances such as drinking
water analysis and cell or tissue-based assays in biological media.
In the latter case, mildly acidic pH condition (e.g., intracellular
microenvironment: pH ∼5ꢀ6 for endosomes and lysosomes)
might be encountered. However, it is well-known that the
silyl ether moiety in SiCouMA is less stable and subjected to
A closer examination of Figures 5a and 6a revealed that upon
complete reaction of SiCouMA species of 8, diblock unimer
solutions at 20 ꢀC exhibit higher emission intensities than that for
diblock micelles at 40 ꢀC. To further probe this, the temperature-
dependent fluorescence emission spectra were collected for 8 in
aqueous solution subjected to complete SiCouMA reaction
(1600 equiv., overnight). We observe the prominent decrease
8788
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
Supporting Information. It was found that the time-dependent
profile of emission intensity changes at pH 5.0 is quite compar-
able to those occurred at pH 7.0. After 1 h incubation in the
absence of Fꢀ ions, ∼3.3-fold and 3.1-fold increase in emission
intensities were observed at pH 5.0 and pH 7.4, respectively. We
also found that at pH 3.0, the hydrolysis reaction is much faster,
exhibiting ∼10.7-fold emission intensity increase after incuba-
tion for duration of 1 h. Thus, for strongly acidic aqueous samples
(pH < 5), though less frequently associated with bioassay
conditions, the preadjusting of solution pH to >5.0 is quite
preferred. Another issue is the thermal stability of SiCouMA
moieties on PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA).
We attempted to heat the THF solution of diblock copolymer
to 50 ꢀC and monitored the evolution of emission intensities at
402 nm (Figure S10, Supporting Information). It was found that
within ∼400 min upon heating, the emission intensity remains
essentially constant, suggesting that SiCouMA moieties on the
diblock copolymer are structurally stable at elevated tempera-
tures. Note that the slight increase in emission intensity occurred
initially should be ascribed to the presence of residual water
traces in THF.
Figure 7. Determination of Fꢀ detection limits for 0.5 g/L aqueous
solution of PEO-b- P(MEO2MA-co-OEGMA-co-SiCouMA) (8, 5.3 μM
SiCouMA moieties; 20 mM phosphate buffer) at (a) 20 and (b) 40 ꢀC,
respectively.
’ CONCLUSIONS
In summary, we report on the fabrication of a novel type of
responsive double hydrophilic block copolymer (DHBC)-based
highly selective and sensitive fluorescence “turn-on” reactive
probes for fluoride ions (Fꢀ) working in purely aqueous media
by exploiting Fꢀ-induced cyclization reaction of nonfluorescent
moieties to induce the formation of fluorescent coumarin
moieties within the thermoresponsive block. Diblock copoly-
mers bearing Fꢀ-reactive moieties (SiCouMA) in the thermo-
responsive block, PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA),
were synthesized, which molecularly dissolve in water at room
temperature and self-assemble into micellar nanoparticles above
the critical micellization temperature (CMT, 33 ꢀC). In the
presence of Fꢀ ions, deprotection of nonfluorescent SiCouMA
moieties followed by spontaneous cyclization reaction leads to
the formation of highly fluorescent coumarin residues (CouMA).
Thus, PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA) diblock
copolymers can serve as highly efficient and selective fluores-
cence “turn-on” reaction probes for Fꢀ ions in aqueous media. In
the range of 0ꢀ1600 equiv of Fꢀ ions, diblock unimers and
micellar solutions at 20 and 40 ꢀC exhibit ∼88-fold and ∼30-fold
increase in fluorescence emission intensity (20 min incubation
time), respectively. The detection limits were determined to be
0.065 and 0.05 ppm for diblock unimers and micelles, respec-
tively. Upon complete transformation of nonfluorescent SiCou-
MA moieties into fluorescent CouMA, the emission intensity of
diblock copolymer solution decreases linearly with temperatures
in the range of 20ꢀ60 ꢀC, suggesting its further application as
fluorometric temperature sensors.
Figure 8. (a) Fluorescence emission spectra and (b) relative fluores-
cence emission intensities at 402 nm recorded for 0.5 g/L aqueous
solution (20 ꢀC) of PEO-b-P(MEO2MA-co-OEGMA-co-SiCouMA) ꢀat
To the best of our knowledge, this work represents the first
example of Fꢀ-reactive polymeric probes working in purely aqueous
media, which are capable of highly sensitive and selective
fluorescent Fꢀ sensing in the form of both unimers and micellar
nanoparticles. We demonstrate that the combination of stimuli-
responsive block copolymers with chemical reaction-based small
molecule fluorescent sensing motifs can offer combined advan-
tages such as water dispersibility, biocompatibility, and the
capability of integration with temperature sensing functions.
One drawback of the current system is that calibration curves
∼20 min after the addition of 100 equiv of Clꢀ, Brꢀ, Iꢀ, CO32‑, NO3
,
HSO4ꢀ, HPO42‑, H2PO4ꢀ, AcOꢀ, and Fꢀ ions (sodium salts in all cases),
respectively.
hydrolysis reaction in acidic media, which is especially fast at low
pH. To further probe the scope of applications, we also examined
the stability of PEO-b-P(MEO2MA-co-OEGMA- co-SiCouMA)
copolymer at pH 5.0 and the results are shown in Figure S9,
8789
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790

Macromolecules
ARTICLE
need to be established prior to sample measurements, partially
due to that the fluorometric sensing is based on intensity changes
of a single emission band. Further works including the fabrication
of responsive polymer-based ratiometric fluorescent Fꢀ probes
are currently underway.
(26) Arimori, S.; Fyles, T. M.; Hibbert, T. G.; James, T. D.; Kociok-
Kohn, G. I.; Davidson, M. G. Chem. Commun. 2004, 1640–1641.
(27) Saxena, A.; Fujiki, M.; Rai, R.; Kim, S. Y.; Kwak, G. Macromol.
Rapid Commun. 2004, 25, 1771–1775.
(28) Melaimi, M.; Gabbai, F. P. J. Am. Chem. Soc. 2005, 127, 9680–9681.
(29) Xu, S.; Chen, K. C.; Tian, H. J. Mater. Chem. 2005, 15, 2676–2680.
(30) Hudson, Z. M.; Wang, S. N. Acc. Chem. Res. 2009, 42, 1584–1596.
(31) Liu, X. Y.; Bai, D. R.; Wang, S. N. Angew. Chem., Int. Ed. 2006,
45, 5475–5478.
’ ASSOCIATED CONTENT
1
S
Supporting Information. Characterization data of H
b
(32) Shirasaka, T.; Akiyama, S.; Tamao, K.; Yamaguchi, S. J. Am.
Chem. Soc. 2002, 124, 8816–8817.
NMR, 13C NMR, GPC, and additional fluorescence spectro-
metry results. This material is available free of charge via the
Internet at http://pubs.acs.org.
(33) Bozdemir, O. A.; Sozmen, F.; Buyukcakir, O.; Guliyev, R.;
Cakmak, Y.; Akkaya, E. U. Org. Lett. 2010, 12, 1400–1403.
(34) Bhalla, V.; Singh, H.; Kumar, M. Org. Lett. 2010, 12, 628–631.
(35) Zhang, J. F.; Lim, C. S.; Bhuniya, S.; Cho, B. R.; Kim, J. S. Org.
Lett. 2011, 13, 1190–1193.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: (S.L.) sliu@ustc.edu.cn; (H.Z.) huizhong@hytc.edu.cn.
(36) Kim, S. Y.; Hong, J. I. Org. Lett. 2007, 9, 3109–3112.
(37) Sokkalingam, P.; Lee, C. H. J. Org. Chem. 2011, 76, 3820–3828.
(38) Zhu, B. C.; Yuan, F.; Li, R. X.; Li, Y. M.; Wei, Q.; Ma, Z. M.; Du,
B.; Zhang, X. L. Chem. Commun. 2011, 47, 7098–7100.
(39) Baker, M. S.; Phillips, S. T. J. Am. Chem. Soc. 2011, 133, 5170–5173.
(40) Yang, X. F. Spectrochim. Acta A 2007, 67, 321–326.
(41) Yang, X. F.; Qi, H. P.; Wang, L. P.; Su, Z.; Wang, G. Talanta
2009, 80, 92–97.
’ ACKNOWLEDGMENT
The financial support from National Natural Scientific Foun-
dation of China (NNSFC) Projects (20874092, 91027026,
20975043, and 51033005) and Fundamental Research Funds for
the Central Universities is gratefully acknowledged.
(42) Yang, X. F.; Ye, S. J.; Bai, Q.; Wang, X. Q. J. Fluoresc. 2007,
17, 81–87.
(43) Li, Y. M.; Zhang, X. L.; Zhu, B. C.; Yan, J. L.; Xu, W. P. Anal. Sci.
2010, 26, 1077–1080.
’ REFERENCES
(44) Zhu, C. Q.; Chen, J. L.; Zheng, H.; Wu, Y. Q.; Xu, J. G. Anal.
Chim. Acta 2005, 539, 311–316.
(45) Hu, R.; Feng, J. A.; Hu, D. H.; Wang, S. Q.; Li, S. Y.; Li, Y.; Yang,
G. Q. Angew. Chem., Int. Ed. 2010, 49, 4915–4918.
(46) Kim, S. Y.; Park, J.; Koh, M.; Park, S. B.; Hong, J. I. Chem.
Commun. 2009, 4735–4737.
(1) Beer, P. D.; Gale, P. A. Angew. Chem., Int. Ed. 2001, 40, 486–516.
(2) Kostyuk, P. G.; Krishtal, O. A.; Pidoplichko, V. I. Nature 1975,
257, 691–693.
(3) Kubik, S. Chem. Soc. Rev. 2009, 38, 585–605.
(4) Langmuir, D. Geochim. Cosmochim. Acta 1978, 42, 547–569.
(5) Schmidtchen, F. P.; Berger, M. Chem. Rev. 1997, 97, 1609–1646.
(6) Kirk, K. L. Biochemistyr of the Halogens and Inorganic Halides;
Plenum Press: New York, 1991; p58.
(47) Li, G.; Gong, W. T.; Ye, J. W.; Lin, Y. A.; Ning, G. L. Tetrahedron
Lett. 2011, 52, 1313–1316.
(48) Padie, C.; Zeitler, K. New J. Chem. 2011, 35, 994–997.
(49) Hu, R.; Feng, J. A.; Hu, D. H.; Wang, S. Q.; Li, Y.; Yang, G. Q.
Angew. Chem., Int. Ed. 2010, 49, 4915–4918.
(50) Liu, T.; Liu, S. Y. Anal. Chem. 2011, 83, 2775–2785.
(51) Hu, J. M.; Li, C. H.; Liu, S. Y. Langmuir 2010, 26, 724–729.
(52) Hu, J. M.; Liu, S. Y. Macromolecules 2010, 43, 8315–8330.
(53) Yoon, J.; Kim, H. N.; Guo, Z. Q.; Zhu, W. H.; Tian, H. Chem.
Soc. Rev. 2011, 40, 79–93.
(54) Yin, J.; Hu, H. B.; Wu, Y. H.; Liu, S. Y. Polym. Chem. 2011,
2, 363–371.
(55) Qu, Y.; Hua, J. L.; Jiang, Y. H.; Tian, H. J. Polym. Sci., Part A:
Polym. Chem. 2009, 47, 1544–1552.
(7) Katruzov, A. N.; Nikitina, T. G.; Moskvin, L. N. J. Anal. Chem.
1998, 53, 173–177.
(8) Frant, M. S.; Ross, J. W. Science 1966, 154, 1553–&.
(9) Hara, H.; Huang, C. C. Anal. Chim. Acta 1997, 338, 141–147.
(10) Bodor, R.; Madajova, V.; Masar, M.; Johnck, M.; Stanislawski,
B.; Kaniansky, D. J. Chromatogra. A 2001, 916, 155–165.
(11) Cametti, M.; Rissanen, K. Chem. Commun. 2009, 2809–2829.
(12) Boiocchi, M.; Del Boca, L.; Gomez, D. E.; Fabbrizzi, L.;
Licchelli, M.; Monzani, E. J. Am. Chem. Soc. 2004, 126, 16507–16514.
(13) Esteban-Gomez, D.; Liechelli, M.; Fabbrizzi, L. J. Org. Chem.
2005, 70, 5717–5720.
(14) Kruger, P. E.; Jensen, P.; Pfeffer, F. M.; Hussey, G. M.;
Gunnlaugsson, T. Tetrahedron Lett. 2003, 44, 8909–8913.
(15) Qu, Y.; Hua, J. L.; Tian, H. Org. Lett. 2010, 12, 3320–3323.
(16) Bok, J. H.; Bartsch, R. A.; Lee, J. Y.; Kim, J. S.; Kim, S. K. Org.
Lett. 2005, 7, 4839–4842.
(56) Hu, J. M.; Li, C. H.; Cui, Y.; Liu, S. Y. Macromol. Rapid Commun.
2011, 32, 610–615.
(57) Hu, J. M.; Zhang, G. Y.; Geng, Y. H.; Liu, S. Y. Macromolecules
2011, DOI: 10.1021/ma201777p.
(58) Kim, T. H.; Swager, T. M. Angew. Chem., Int. Ed. 2003,
42, 4803–4806.
(59) Thomas, S. W.; Joly, G. D.; Swager, T. M. Chem. Rev. 2007,
(17) Liu, B.; Tian, H. J. Mater. Chem. 2005, 15, 2681–2686.
(18) Cho, E. J.; Moon, J. W.; Ko, S. W.; Lee, J. Y.; Kim, S. K.; Yoon, J.;
Nam, K. C. J. Am. Chem. Soc. 2003, 125, 12376–12377.
(19) Mizuno, T.; Wei, W. H.; Eller, L. R.; Sessler, J. L. J. Am. Chem.
Soc. 2002, 124, 1134–1135.
(20) Peng, X. J.; Wu, Y. K.; Fan, J. L.; Tian, M. Z.; Han, K. L. J. Org.
Chem. 2005, 70, 10524–10531.
(21) He, X. M.; Hu, S. Z.; Liu, K.; Guo, Y.; Xu, J.; Shao, S. J. Org. Lett.
2006, 8, 333–336.
(22) Wang, Q. G.; Ding, Y. B.; Li, X.; Zhu, W. H.; Xie, Y. S. Chem.
107, 1339–1386.
(60) Cai, Y. L.; Armes, S. P. Macromolecules 2005, 38, 271–279.
(61) Hoth, A.; Lutz, J. F. Macromolecules 2006, 39, 893–896.
(62) Han, S.; Ishizone, T.; Hagiwara, M. Macromolecules 2003,
36, 8312–8319.
(63) Amir, R. J.; Albertazzi, L.; Willis, J.; Khan, A.; Kang, T.; Hawker,
C. J. Angew. Chem., Int. Ed. 2011, 50, 3425–3429.
(64) Wang, B. Y.; Guan, X. L.; Hu, Y. L.; Su, Z. X. Polym. Adv.
Technol. 2007, 18, 529–534.
Commun. 2010, 46, 3669–3671.
(23) He, X. M.; Yam, V. W. W. Org. Lett. 2011, 13, 2172–2175.
(24) Yang, Z. P.; Zhang, K.; Gong, F. B.; Li, S. Y.; Chen, J.; Ma, J. S.;
Sobenina, L. N.; Mikhaleva, A. I.; Trofimov, B. A.; Yang, G. Q.
J. Photochem. Photobiol. A—Chem. 2011, 217, 29–34.
(25) Kim, S. K.; Yoon, J. Chem. Commun. 2002, 770–771.
8790
dx.doi.org/10.1021/ma2018588 |Macromolecules 2011, 44, 8780–8790