Thermoresponsive core cross-linked micelles for selective ratiometric fluorescent detection of Hg2+ ions

Article abstract of DOI:10.1021/la104911r

We report on the fabrication of core cross-linked (CCL) micelles possessing thermoresponsive cores and their application as sensitive and selective ratiometric Hg²⁺ probes with thermo-tunable detection efficiency. Well-defined double hydrophili

Full text of DOI:10.1021/la104911r

ARTICLE
pubs.acs.org/Langmuir
Thermoresponsive Core Cross-Linked Micelles for Selective
Ratiometric Fluorescent Detection of Hg2þ Ions
Xuejuan Wan, Tao Liu, 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
S Supporting Information
b
ABSTRACT: We report on the fabrication of core cross-linked (CCL)
micelles possessing thermoresponsive cores and their application as sensi-
tive and selective ratiometric Hg2þ probes with thermo-tunable detection
efficiency. Well-defined double hydrophilic block copolymer (DHBC)
bearing naphthalimide-based Hg2þ-reactive moieties (NUMA, 4), PEO-b-
P(NIPAM-co-NAS-co-NUMA), was synthesized via reversible addition-
fragmentation chain transfer (RAFT) polymerization, where PEO, NIPAM,
and NAS represent poly(ethylene oxide), N-isopropylacrylamide, and N-
acryloxysuccinimide. At 25 ꢀC, PEO-b-P(NIPAM-co-NAS-co-NUMA) un-
imers in aqueous solution can act as ratiometric Hg2þ probes with a
detection limit of ∼10.1 nM. After core cross-linking of the micellar
nanoparticles formed at elevated temperatures, structurally stable CCL
micelles with well-solvated PEO coronas and thermoresponsive cores
embedded with Hg2þ-reactive NUMA moieties were obtained. Upon
Hg2þ addition, the aqueous dispersion of CCL micelles exhibit a colori-
metric transition from yellowish to colorless and a fluorometric emission
transition from green to bright blue. Moreover, Hg2þ detection limits of
CCL micelles were considerably enhanced to 3.0 and 1.8 nM at 25 and 40 ꢀC, when the thermoresponsive cores are at their swollen
and collapsed state, respectively. The high selectivity of CCL micelles to Hg2þ over other common cations was also demonstrated.
Furthermore, in vitro studies revealed that CCL micelles can effectively enter into living cells and sensitively respond to the presence
of Hg2þ ions via the change of fluorescence emission color. This work represents the first example of DHBC-based CCL micelle
acting as highly selective and sensitive ratiometric metal ion probes. The structural stability, water dispersibility, biocompatibility,
and most importantly the thermo-tunable detection sensitivity of this novel type of CCL micelle-based sensing systems augur well
for their future applications as multifunctional nanocarriers for drug delivery, sensing, imaging, and diagnosis.
’ INTRODUCTION
boron dipyrromethene dye (BODIPY) as the FRET donor and
leuco-rhodamine derivative as the acceptor.10 Taking advantage of
the highly efficient ring-opening reaction of nonfluorescent rhoda-
mine derivative induced by Hg2þ, ratiometric Hg2þ detection based
on the “off-on” switching of FRET process can be achieved. The
second strategy involves the utilization of an internal standard in
combination with Hg2þ-responsive fluorescent moieties.39,44 In a
recent example, Zhang et al.39 reported a ratiometric naphthali-
mide-porphyrin hybrid probe for Hg2þ, and the two Hg2þ-sensitive
fluorophores possess almost independent fluorescent emission due
to large differences (∼125 nm) in their maximum emission wave-
lengths and quite comparable excitation bands. This can effectively
eliminate the FRET process between these two fluorophores. The
third strategy for the design of ratiometric Hg2þ sensors is based on
the intramolecular charge transfer (ICT) process by utilizing only
The sensitive and selective detection of mercury pollutants
both in vivo and in vitro is quite crucial considering their bioaccu-
mulation, long residence, and permanent damage to central nervous
and endocrine systems.1,2 During the past decades, a variety of
fluorometric and colorimetric mercury sensors have been fabricated
from small organic molecules,3-12 biomolecules,13-19 conjugated
polymers,20-24 nanoparticles,13,15,25-33 and polymeric assemb-
lies.34-37 Fluorometric Hg2þ probes are based on changes in either
the relative fluorescence emission intensity or intensity ratio (if mul-
tiple emission bands are present) induced by the presence of Hg2þ
ions. Compared to the former, the latter reports ratiometric values
and can effectively eliminate the background interference and the
fluctuation of detection conditions.7,10,26,38-47
Three main strategies have been employed to construct ratiomet-
ric fluorometric Hg2þ sensors. The first one relies on the utilization
of fluorescence resonance energy transfer (FRET) principle.10,38,43,46
Recently, Qian and his co-workers reported an elegant example of
FRET-based small molecule ratiometric Hg2þ sensor by employing
Received: December 10, 2010
Revised:
February 11, 2011
Published: March 02, 2011
r
2011 American Chemical Society
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one type of fluorophore, the fluorescence emission band of which
exhibits a remarkable shift upon binding with Hg2þ ions or reacting
in the presence of Hg2þ ions.7,26,41,45,48 In this context, Tian et al.7
reported an Hg2þ-reactive ratiometric chemosensor based on
naphthalimide phenylthiourea derivatives. Upon addition of Hg2þ
ions, considerable ICT-induced shift of the fluorescence emission
band occurs as the electron-donating amine moiety was replaced by
less electron-rich guanidine functionality. Recently, we covalently
attached a Hg2þ-sensing naphthalimide derivative into thermore-
sponsive poly(N-isopropylacrylamide) (PNIPAM) nanogels and
fabricated polymeric nanoparticle-based ratiometric Hg2þ sensors
with thermo-tunable detection sensitivity.26
Scheme 1. Schematic Illustrationsa
Compared to small molecule fluorometric probes, sensing
systems based on polymeric nanoparticles and assemblies can
offer additional advantages such as improved water solubility,
longer in vivo circulation times, and larger accumulation capacity
due to the enhanced permeability and retention (EPR) effect;
most importantly, the integration of multifunctions such as
sensing, imaging, and therapeutics can also be achieved. In the
past few years, our research work has mainly focused on the
synthesis and self-assembly of stimuli-responsive amphiphilic
and double hydrophilic block copolymers (DHBCs) with vary-
ing chain topologies.49-53 To explore the applications of poly-
meric assemblies in sensing and detection, we recently reported
the synthesis of well-defined DHBCs bearing rhodamine B
derivatives (RhBHA) in the thermoresponsive block, poly-
(ethylene oxide)-b-P(NIPAM- co-RhBHA), PEO-b-P(NIPAM-
co-RhBHA), which can serve as multifunctional sensors to pH,
temperature, and Hg2þ ions.35,54 Most importantly, the detec-
tion sensitivity to Hg2þ and pH could be dramatically enhanced
at elevated temperature due to thermo-induced formation of
micelles possessing hydrophobic cores. However, the detection
protocol is mainly based on the changes in relative fluorescence
emission intensity, and most importantly, micelles of PEO-b-
P(NIPAM-co-RhBHA) are not stable at temperatures below the
critical micellization temperature (CMT) and or at polymer
concentrations below the critical micellization concentration
(CMC). This will considerably limit their practical applications
as polymeric assemblies-based sensing systems.54
In this context, several approaches such as core cross-linking
(CCL) and shell cross-linking (SCL) have been proposed to
enhance the structural stability of polymeric micelles.55-60
Inspired by the work of McCormick et al.,57 we previously
reported the fabrication of reversible CCL micelles from PEO-
b-P(NIPAM-co-NAS), which bears reactive acryloxysuccinimide
(NAS) moieties.61 Upon thermo-induced micellization, core
cross-linking can be conveniently achieved by reaction of NAS
residues in the micellar cores with a difunctional cross-linker,
cystamine. In this work, we further attempted to utilize structu-
rally stable CCL micelles with thermoresponsive cores as a
platform to construct novel sensing systems (Scheme 1). We
covalently attached Hg2þ-reactive moieties (NUMA) into the
thermoresponsive block of PEO-b-P(NIPAM-co-NAS), and the
obtained PEO-b- P(NIPAM-co-NAS-co-NUMA) DHBC can
self-assemble into core-shell nanoparticles possessing thermo-
responsive PNIPAM cores which can be cross-linked. We then
determined and compared the Hg2þ-sensing capability of PEO-
b-P(NIPAM-co-NAS-co-NUMA) unimers and CCL micelles at
temperatures below and above the critical phase transition
temperature. It was found that the aqueous dispersion of CCL
micelles exhibits a colorimetric transition from yellowish to
colorless and a fluorometric emission transition from green to
a (a) Preparation of core cross-linked (CCL) micelles with thermo-
responsive cores from PEO-b-P(NIPAM-co-NAS-co-NUMA) diblock
copolymer in aqueous solution and the Hg2þ-reactive mechanism of
NUMA moieties. (b) Hg2þ-sensing capability of CCL micelles with
improved detection sensitivity at elevated temperatures.
bright blue upon addition of Hg2þ ions. Most importantly, the
detection sensitivity to Hg2þ ions (∼3.0 nM at 25 ꢀC, ∼1.8 nM
at 40 ꢀC) of CCL micelles constructed from PEO-b-P(NIPAM-
co-NAS-co- NUMA) can be considerably enhanced compared to
that at unimer state (∼10.1 nM). The fluorescence imaging assay
of Hg2þ ions in living cells was also investigated. As far as we
know, this represents the first report of responsive DHBC-based
CCL micelles which can act as highly selective and sensitive
ratiometric probes for Hg2þ ions.
’ EXPERIMENTAL SECTION
Materials. N-Isopropylacrylamide (NIPAM, 97%, Tokyo Kasei Kagyo
Co.) was purified by recrystallization from a mixture of benzene and n-
hexane (1/3, v/v). Monohydroxy-capped poly(ethylene oxide), (PEO113
-
OH, Mn = 5100, Mw/Mn = 1.10, mean degree of polymerization, DP, is 113)
was purchased from Aldrich and used as received. 4-Bromo-1,8-naphthalic
anhydride and methacrylic anhydride were purchased from Aldrich and used
without further purification. Fetal bovine serum (FBS), penicillin, strepto-
mycin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased
from GIBCO and used as received. 2,20-Azobisisobutyronitrile (AIBN, 98%,
Fluka) was recrystallized from 95% ethanol. N-Hydroxysuccinimide (NHS,
97%, Aldrich) was recrystallized from toluene prior to use. N,N0-Dicyclohex-
ylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), potassium thio-
cyanate (KSCN), 2-aminoethanol, 1,2-ethylenediamine, benzoyl chloride,
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Scheme 2. Schematic Illustration for the Synthesis of 1,8-
Naphthalimide-Based Hg2þ-Reactive Polymerizable Mono-
mer (NUMA, 4)
solid (0.37 g, 69.7% yield). 1H NMR (DMSO-d6, δ, ppm; Figure S2b):
8.64, 8.38, 8.21, 7.64, 6.93 (5H, naphthalimide-H), 7.83, 7.55, 7.44
(5H, Ar-H), 5.86, 5.13 (3H, CH2dCH(CH3)-), 4.71 (2H, -
CH2CH2OCdO), 4.31 (2H, -CH2CH2OCdO), 4.06 (2H, -
NHCH2CH2NHCdS), 3.97 (2H, -NHCH2CH2NHCdS), 2.13
(3H, CH2dCH(CH3)-).
Synthesis of PEO-b-P(NIPAM-co-NAS-co-NUMA) Diblock Copoly-
mer. PEO macroRAFT agent was synthesized via the DCC-mediated
esterification reaction of PEO113-OH with DDMATC according to
literature procedures.35 PEO-b-P(NIPAM-co-NAS-co-NUMA) diblock
copolymer was prepared via RAFT copolymerization according to
similar procedures we reported previously.61 Typically, PEO113 macro-
RAFT agent (0.54 g, 0.1 mmol), NIPAM (2.26 g, 20 mmol), NAS (0.38 g,
2.2 mmol), NUMA (57 mg, 0.11 mmol), and AIBN (3 mg, 0.02 mmol)
were charged into a glass ampule containing 4 mL of 1,4-dioxane. The
ampule was degassed through three freeze-thaw cycles, sealed under
vacuum, and kept in an oil bath thermostatted at 70 ꢀC to start the
polymerization. After 2 h, theglass ampule was soakedinto liquidnitrogen
to quench the polymerization. The reaction mixture was diluted with
THF and precipitated into an excess of diethyl ether. The sediments were
collected and dried in a vacuum oven overnight at room temperature to
obtain a yellowish green solid at a yield of 59%. The molecular weight and
molecular weight distribution of PEO-b-P(NIPAM-co-NAS-co-NUMA)
diblock copolymer were determined by GPC using THF as eluent,
revealing an Mn of 18 200 and an Mw/Mn of 1.19 (Figure S3). The
overall DP of P(NIPAM-co-NAS-co-NUMA) block and the NAS content
and all other reagents were purchased from Shanghai Chemical Reagent Co.
and used as received. Benzoyl isothiocyanate,62 N-acryloxysuccinimide
(NAS),63 and S-1-dodecyl-S0-(R,R0-dimethyl-R00-acetic acid)trithiocarbo-
nate (DDMATC)64 were synthesized according to the literature procedures.
Water was deionized with a Milli-Q SP reagent water system (Millipore) to a
specific resistivity of 18.4 MΩ cm.
Sample Preparation. General approaches employed for the pre-
paration of 1,8-naphthalimide based Hg2þ-sensing fluorescent mono-
mer, NUMA (4), are shown in Scheme 2.
Preparation of 1,8-Naphthalimide-Based Hg2þ-Reactive Fluores-
cent Monomer (NUMA). The reaction of 4-bromo-1,8-naphthalic
anhydride (2.77 g, 0.01 mol) and 2-aminoethanol (0.61 g, 0.01 mol)
was conducted for 4 h in boiling 1,4-dioxane (50 mL) under magnetic
stirring. After cooling to room temperature, the yellowish sediments
were collected by filtration and then dried overnight at room tempera-
ture in a vacuum oven to give 1 (3.03 g, yield: 94.7%). 1H NMR (CDCl3,
δ, ppm; Figure S1a): 8.64, 8.56, 8.40, 8.03, 7.85 (5H, naphthalimide-H),
4.45 (2H, -CH2CH2OH), 3.98 (2H, -CH2CH2OH).
A mixture of compound 1 (2.24 g, 7.0 mmol) and 1,2-ethylenediamine
(12.6 g, 0.21 mol) in 80 mL 2-methoxylethanol was heated to reflux and
stirredfor3 h. Aftercoolingtoroomtemperature, the reactionmixture was
concentrated to 30 mL and poured into 200 mL water. Sediments were
collected and then purified by recrystallization from chlorobenzene to give
2 as an orange solid (1.36 g, yield: 64.9%). 1H NMR (DMSO-d6, δ, ppm;
Figure S1b): 8.64, 8.38, 8.18, 7.61, 6.74 (5H, naphthalimide-H), 4.06
(2H, -CH2CH2OH), 3.52 (2H, -CH2CH2OH), 2.81 (2H, -NHCH2
CH2NH2).
Compound 2 (0.90 g, 3 mmol) was dissolved in 30 mL dry acetone,
and then freshly synthesized benzoyl isothiocyanate (0.49 g, 3 mmol) in
20 mL dry acetone was added dropwise over 1 h. The mixture was heated
to 45 ꢀC and allowed to stir for another 4 h. After cooling to room
temperature, the sediments were collected by filtration and washed with
dry acetone three times. The crude product was further purified by
chromatography using ethyl acetate/CH2Cl2 as the eluent. After drying
in a vacuum oven at room temperature overnight, 3 was obtained as an
orange solid (0.87 g, yield: 62.7%). 1H NMR (DMSO-d6, δ, ppm; Figure
S2a): 8.65, 8.48, 8.33, 7.95, 6.84 (5H, naphthalimide-H), 7.42-7.66
(5H, Ar-H), 4.26 (2H, -CH2CH2OH), 4.18 (2H, -CH2CH2OH),
3.74 (4H, -NHCH2CH2NH-).
A mixture of compound 3 (0.46 g, 1 mmol) and methacrylic
anhydride (0.18 mL, 1.2 mmol) in 30 mL dry pyridine was stirred
overnight at room temperature. The reaction mixture was then diluted
with ethyl acetate and washed three times with saturated aqueous
solution of NaHCO3. The organic layer was collected and dried over
anhydrous MgSO4. After filtration, the filtrate was evaporated to dryness
under reduced pressure. The crude product was further purified by
chromatography using CH2Cl2 as eluent. After drying in a vacuum oven
at room temperature overnight, NUMA (4) was obtained as an orange
1
were determined to be 110 and 16.0 mol %, respectively, by H NMR
analysis in CDCl3 (Figure S4); thus, the copolymer was denoted as PEO113
-
b-P(NIPAM0.84-co-NAS0.16-co-NUMA)110. As the feed ratio of NUMA is
quite low (∼0.5 mol %), its molar content (∼0.4 mol %) was determined by
fluorescence measurements in methanol against a standard calibration curve.
Synthesis of CCL Micelles. 0.60 g PEO113-b-P(NIPAM0.84-co-
NAS0.16-co-NUMA)110 was dissolved in 40 mL deionized water at
25 ꢀC, and then heated to 40 ꢀC. A bluish tinge characteristic of
colloidal aggregates appeared upon heating, indicating the formation of
micellar nanoparticles. After equilibration at 40 ꢀC for 30 min, 8.1 mL
aqueous solution of cystamine dihydrochloride (adjusted to pH 7, 5.0 g/L,
40 ꢀC) was injected. The cystamine/NAS molar ratio was kept at 1:2. The
reaction mixture was stirred for 5 h at 40 ꢀC. After the mixture was cooled
to 25 ꢀC, the bluish tinge characteristic of micellar nanoparticles persisted,
suggesting the successful core cross-linking.
Cell Culture and Fluorescence Imaging of Hg2þ Ions in Living Cells.
HeLa cells were cultured in Dulbecco’s modified Eagle medium
(DMEM) supplemented with 10% FBS, penicillin (100 units/mL),
and streptomycin (100 g/L) at 37 ꢀC in a CO2/air (5:95) incubator for 2
d. For fluorescence imaging, cells were first transferred to DMEM
containing 1% FBS, and CCL micelles of PEO113-b-P(NIPAM0.84-co-
NAS0.16-co-NUMA)110 (0.45 g/L, 10.0 μM NUMA residues) at 37 ꢀC in
a CO2/air (5:95) incubator for 4 h; Hg2þ ions were then added to target
a final concentration of 50.0 μM, and the mixture was further incubated
for 30 min.
Characterization. All nuclear magnetic resonance (NMR) spectra
were recorded on a Bruker 300 MHz spectrometer using CDCl3 or
DMSO-d6 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.
UV-vis spectra were acquired on a Unico UV/vis 2802PCS spectro-
photometer. The transmittance of the aqueous solutions was acquired at
a wavelength of 600 nm. A commercial spectrometer (ALV/DLS/SLS-
5022F) equipped with a multitau digital time correlator (ALV5000) and
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a cylindrical 22 mW Uniphase He-Ne laser (λ0 = 632 nm) as the light
source was employed for dynamic laser light scattering (LLS) measure-
ments. Scattered light was collected at a fixed angle of 90ꢀ for duration of
∼5 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. The fluorescence images of HeLa cells
incubated with CCL micelles in the presence and absence of Hg2þ ions
were acquired on a Nikon Eclipse TE2000-U inverted microscopy.
’ RESULTS AND DISCUSSION
Well-defined DHBCs bearing Hg2þ-sensing moieties in the
thermoresponsive block, PEO-b-P(NIPAM-co-NAS-co-NUMA),
was synthesized via RAFT copolymerization by utilizing PEO-based
macroRAFT agent.65,66 The obtained diblock copolymer molecu-
larly dissolves in aqueous solution at room temperature and self-
assembles into core-shell nanoparticles consisting of thermore-
sponsive P(NIPAM-co-NAS-co-NUMA) cores and well-solvated
PEO coronas. Upon core cross-linking at elevated temperatures,
structurally stable CCL micelles was obtained, which can further
serve as effective colorimetric and fluorometric probes to Hg2þ with
thermo-tunable detection efficiency (Scheme 1).
Figure 1. Temperature dependence of the optical transmittance at
800 nm obtained for 1.0 g/L aqueous solution of PEO113-b-P-
(NIPAM0.84-co-NAS0.16-co-NUMA)110 diblock copolymer before and
after core cross-linking.
transmittance curve (Figure 1). To enhance the structural
stability of micelles of PEO-b-P(NIPAM-co-NAS-co-NUMA)
formed at elevated temperatures, core cross-linking with cysta-
mine was further conducted. After core cross-linking at 40 ꢀC,
dynamic LLS measurements revealed a ÆRhæ of ∼37 nm for CCL
micelles at 25 ꢀC. This indicates the successful fabrication of
CCL micelles possessing thermoresponsive cores; otherwise,
micelles will dissociate into unimers at temperatures below the
CMT (∼28 ꢀC).61 We can also note from Figure S5 that, upon
heating from 20 to 40 ꢀC, ÆRhæ of CCL micelles decreases from
37 to 27 nm due to the thermo-induced collapse of cross-linked
cores. Moreover, compared to that before cross-linking, the
temperature-dependent optical transmittance of CCL micelles
only exhibits slight changes with increasing temperatures
(Figure 1).
Synthesis of 1,8-Naphthalimide-Based Hg2þ-Sensing
Monomer (NUMA, 4), PEO-b-P(NIPAM-co-NAS-co-NUMA)
DHBC, and Fabrication of CCL Micelles. The fluorescent
monomer capable of fluorescent Hg2þ-sensing, NUMA (4),
was synthesized in four steps as illustrated in Scheme 2. The
reaction of 4-bromo-1,8-naphthalic anhydride with 2-aminoetha-
nol afforded 1 at a relatively high yield. Its chemical structure was
confirmed by 1H NMR analysis, as evidenced by the appearance
of resonance signals at 3.9 ppm (peak b) and 4.4 ppm (peak a)
(Figure S1), which should be assigned to methylene protons
adjacent to the amide functionality. Compound 2 was synthe-
sized by the substitution reaction of 1 in the presence of 1,2-
ethylenediamine. The 1H NMR spectrum of 2 is also shown in
Figure S1, together with the peak assignments. We can clearly
observe the appearance of a new resonance signal at 2.9 ppm,
which can be ascribed to methylene protons adjacent to the
terminal primary amine group (peak i).
Compound 2 was then reacted with benzoyl isothiocyanate,
and the subsequent esterification reaction of 3 with methacrylic
anhydride finally afforded the target Hg2þ-sensing monomer 4.
1H NMR spectra of 3 and 4 are shown in Figure S2, together with
the peak assignments. Compared to the NMR spectrum of 2, new
resonance signals characteristic of phenyl protons of 4 appeared
in the range 7.4-7.6 ppm (peaks j, k, and l), accompanied by the
presence of methacrylate double bond signals at 5.1 and 5.8 ppm.
This indicates the successful preparation of Hg2þ-sensing mono-
mer 4.
CCL Micelles of PEO-b-P(NIPAM-co-NAS-co-NUMA) as
Selective Colorimetric and Fluorometric Probes for Hg2þ
Ions. Specially designed small molecules containing thiourea
moieties have been frequently employed to construct Hg2þ
-
reactive colorimetric and fluorometric probes.7,8,67-71 In 2007,
Kim et al.71 developed Hg2þ probes based on Hg2þ-triggered
intramolecular guanylation of the reaction product from phenyl
isothiocyanate and rhodamine 6G-ethylenediamine derivative.
The guanylation reaction leads to the ring-opening of rhodamine
6G moiety initially in the spirolactam form, which is nonfluor-
escent. Thus, Hg2þ-triggered “off-on” switching of fluorescence
emission can be achieved. However, the quantitative determina-
tion of Hg2þ can be only achieved through changes in relative
fluorescence intensities. In another notable example, Tian et al.7
synthesized small molecule probes from phenyl isothiocyanate
and 1,8-naphthalimide-ethylenediamine derivative. In this case,
Hg2þ-triggered guanylation reaction results in the replacement
of electron-donating amine substituent on naphthalimide by the
less electron-rich guanidine moiety. Thus, considerable change in
the extent of ICT leads to a dramatic shift in the fluorescence
emission band and, ratiometric quantification of Hg2þ ions can
be achieved. However, this novel type of small molecule probe is
subjected to poor water solubility, which partially limits their
practical applications. In a previous report, we synthesized a
Hg2þ-sensing polymerizable monomer by the reaction of acry-
loyl isothiocyanate with 1,8-naphthalimide-ethylenediamine de-
rivative (NPTUA).26 Thermoresponsive nanogels covalently
Well-defined DHBCs bearing Hg2þ-sensing moieties in the
thermoresponsive block, PEO-b-P(NIPAM-co-NAS-co-NUMA),
was synthesized via RAFT copolymerization following similar proto-
cols previously employed for the synthesis of PEO-b-P(NIPAM-co-
NAS) diblock copolymer.61 As expected, PEO-b-P(NIPAM-co-NAS-
co-NUMA) molecularly dissolves in aqueous solution as unimers with
an intensity-average hydrodynamic radius, ÆRhæ, of 5-6 nm, and self-
assembles at 40 ꢀC into micelles with ÆRhæ of ∼30 nm (Figure S5).
The critical micellization temperature, CMT, was determined to
be ∼28 ꢀC from the intercept of temperature-dependent optical
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embedded with NPTUA were then synthesized and can act as
selective Hg2þ-reactive colorimetric and fluorometric probes.
Most importantly, we found that, at temperatures higher than the
phase transition temperatures, the detection limit of Hg2þ ions
can be considerably enhanced because collapsed nanogels pro-
vide a more hydrophobic microenvironment for the fluorescent
dye and hence exhibit higher fluorescence quantum yield. In this
work, we aim to establish that structurally stable CCL micelles
can be employed as a platform to construct efficient Hg2þ
-
sensing systems (Scheme 1), so that multifunctional polymeric
nanocarriers capable of sensing, imaging, and delivery of ther-
apeutic agents can be fabricated.
For NUMA-labeled PEO-b-P(NIPAM-co-NAS-co-NUMA)
diblock unimers in aqueous solution at room temperature,
UV-vis absorption spectra upon gradual addition of Hg2þ ions
were recorded (Figure S6). As the concentration of Hg2þ ions
increased, the absorption band at 435 nm significantly decreased
along with the growth of a new absorption band at ∼350 nm.
This can be ascribed to Hg2þ ion-triggered intramolecular
guanylation reaction (Scheme 1a). The aqueous solution also
exhibits a colorimetric transition from yellowish to colorless due
to the shift of absorption band into the UV region. From Figure
S6, we can also observe the almost linear relationship between
A0/(A - A0) and 1/[Hg2þ] in the range 0-1.0 equiv Hg2þ ion
relative to that of NUMA residues, where A0 and A are
absorbance intensities at 360 nm in the absence and presence
of Hg2þ ions. This indicates that Hg2þ ion-triggered intramole-
cular guanylation reaction occurs in a stoichiometric manner. As
an isoabsorptive point at 395 nm can be obtained from Figure S6,
in subsequent sections, all fluorescence spectra were acquired at
an excitation wavelength of 395 nm.
Figure 2. (a) Fluorescence emission spectra and (b) fluorescence
intensity ratio changes (I485/I532) recorded for 0.045 g/L aqueous
solution (1.0 μM NUMA residues) of PEO113-b-P(NIPAM0.84-co-
NAS0.16-co-NUMA)110 upon gradual addition of Hg2þ (0-1.5 equiv)
at 25 ꢀC. The spectra were recorded 20 min after Hg2þ addition (pH 7;
λex = 395 nm; slit widths: Ex = 5 nm, Em = 5 nm).
In the absence of Hg2þ ions, the aqueous solution of PEO-b-
P(NIPAM-co-NAS-co-NUMA) exhibits intense green fluores-
cence emission centered at ∼532 nm, indicating that the
copolymerization of NUMA into the thermoresponsive PNI-
PAM block does not affect the emission properties of NUMA
(Figure 2). As the concentration of Hg2þ ions increased, the
intramolecular guanylation reaction afforded naphthalimide
derivatives exhibiting bright blue fluorescence emission at
∼485 nm. It has been well-established that the variation of
electron-donating substituent at the 4-position of 1,8-naphtha-
limide can dramatically affect its fluorescence emission pro-
perties.7,48,72 In the current case, the electron-rich secondary
amine was replaced with a guanidine moiety which possesses
much less electron-donating capability, and this led to a dra-
matic blue shift in the fluorescence emission.
The fluorometric titration spectra of PEO-b-P(NIPAM-co-
NAS-co-NUMA) aqueous solution at 25 ꢀC shown in Figure 2
indicated that, upon the addition of Hg2þ ions, the fluores-
cence intensity ratio (I485/I532) increased from 0.35 in the
absence of Hg2þ to 1.92 in the presence of 1.5 equiv of Hg2þ
ions, i.e., a ∼5.5-fold increase can be achieved. Note that the
most prominent changes in emission intensity ratios occurred
within the range 0-1.0 equiv Hg2þ ions, which agrees quite
well with those shown in Figure S6. If the detection limit is
defined as the Hg2þ concentration at which 10% change
in fluorescence emission intensity ratio relative to the
control can be observed, the detection limit of Hg2þ ions
was determined to be 10.1 nM.
b-P(NIPAM-co-NAS-co-NUMA) diblock copolymer, its
thermo-induced micellization above the CMT will lead to
the formation of micelles with hydrophobic domains em-
bedded with NUMA dyes. The temperature-dependent fluor-
escence emission spectra of PEO-b-P(NIPAM-co-NAS-co-
NUMA) aqueous solution in the absence of Hg2þ was then
investigated (Figure 3). The fluorescence emission intensity
exhibited a ∼3.1-fold enhancement in the temperature range
20-42 ꢀC, accompanied with a slight blue shift from 532 to
523 nm.
Taking advantage of the remarkable reactivity of NAS moieties
toward primary amines,57 the hydrophobic P(NIPAM-co-NAS-
co-NUMA) core of micelles formed at elevated temperatures was
cross-linked with cystamine to afford structurally stable CCL
micelles. The fluorescence emission spectra of CCL micelles in
aqueous solution at 40 ꢀC upon addition of increasing amount of
Hg2þ ions are shown in Figure 4. Note that the micellar cores are
in their collapsed state at this temperature. A new fluorescence
emission band can be clearly observed at ∼468 nm upon gradual
addition of Hg2þ ions, and the intensity ratio, I468/I523, increased
from 0.125 to 2.80 in the same range, i.e., ∼22-fold increase of
emission intensity ratio can be achieved. Most importantly, the
Hg2þ detection limit of CCL micelles at 40 ꢀC was determined to
be 1.8 nM, as compared to the detection limit of 10.1 nM for
diblock unimers at 25 ꢀC.
It is well-established that the quantum yield of certain
fluorophores will be greatly enhanced when they are located
in a more hydrophobic microenvironment.73-75 As for PEO-
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Figure 4. (a) Fluorescence emission spectra and (b) fluorescence
intensity ratio changes (I468/I523) recorded for 0.045 g/L aqueous
solution of CCL micelles (1.0 μM NUMA residues) upon gradual
addition of Hg2þ (0-1.5 equiv) at 40 ꢀC. The spectra were obtained
20 min after Hg2þ addition (pH 7; λex = 395 nm; slit widths: Ex = 5 nm,
Em = 5 nm).
Figure 3. (a) Fluorescence emission spectra and (b) variation of
relative fluorescence intensity recorded for 0.045 g/L aqueous solution
(1.0 μM NUMA residues) of PEO113-b-P(NIPAM0.84-co-NAS0.16-co-
NUMA)110 (pH 7; λex = 395 nm; slit widths: Ex = 5 nm, Em = 5 nm) in
the temperature range 20-42 ꢀC.
For CCL micelles at 25 ꢀC, NUMA moieties are located in a
hydrophilic environment. In the Hg2þ concentration range
0-1.5 equiv relative to NUMA residues, the emission intensity
ratio, I468/I523, increased from 0.165 to 2.4 (Figure 5). Note that,
in the same Hg2þ concentration range, I468/I523 increased ∼22-
fold at 40 ꢀC, which is larger than that at 25 ꢀC (15-fold).
Concomitantly, the detection limit of CCL micelles at 25 ꢀC is
∼3.0 nM. The enhancement of detection limit for CCL micelles
relative to that of diblock unimers at 25 ꢀC, though in both cases,
NUMA residues are in a hydrophilic microenvironment, that can
be tentatively ascribed to the accumulation of NUMA dyes
within the cores of CCL micelles. Thus, Hg2þ might have a
higher probability to encounter and react with NUMA moieties,
even at quite low concentrations. Moreover, the polarity of
the cross-linked core might also be different from that of the
P(NIPAM-co-NAS-co-NUMA) diblock unimer. In general, the
above results successfully manifested that the structural integrity
of CCL micelles can positively contribute to the detection
performance. The changes of relative fluorescence intensities,
I/I0, of the emission band associated with the Hg2þ-triggered
product can also be employed for the quantitative determination
of Hg2þ ions. Figure 6 shows the Hg2þ concentration-dependent
emission intensities of diblock unimers at 25 ꢀC (485 nm) and
CCL micelles at 25 and 45 ꢀC (468 nm). In all three cases, the
Hg2þ detection limits were determined to be 16.7 nM, 4.8 nM,
and 3.8 nM, respectively.
The detection selectivity of CCL micelles fabricated from
PEO-b-P(NIPAM-co-NAS-co-NUMA) toward Hg2þ ions over
other common cations such as Naþ, Kþ, Pb2þ, Cu2þ, Agþ, Zn2þ
,
Fe2þ, Fe3þ, and Hg2þ was also investigated (Figures 7 and 8).
Among all the cations used in the measurements, only Hg2þ
exhibits apparent colorimetric and fluorometric changes. The
presence of Hg2þ ions lead to a colorimetric transition from
yellowish to colorless and a fluorometric transition from
green to bright, which can even be discerned by the naked
eye (Figure 8). It is worth noting that, at extended time
periods, Agþ can also trigger the intramolecular guanylation
reaction of NUMA residues, but it takes a much longer time
(∼20 h) to complete. Moreover, the existence of Cu2þ ions
exhibits no interference for the measurement, demonstrat-
ing that the CCL micelle-based detection system possesses
promising advantages in discriminating among Agþ, Hg2þ
and Cu2þ ions.
,
Finally, we investigated the Hg2þ sensing capacity of CCL
micelles in living cells. As shown in Figure 9a, HeLa cells
cultured in the presence of 0.45 g/L CCL micelles exhibit
green fluorescence, indicating that CCL micelles can effec-
tively enter into cells. When the above culture mixture
was supplemented with 5.0 equiv Hg2þ (relative to NUMA
residues) for 30 min, a distinctive fluorometric transition from
green to blue can be clearly observed by fluorescence micro-
scopy (Figure 9b). This preliminary experiment indicates that
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Figure 7. (a) Fluorescence emission spectra and (b) fluorescence
intensity ratio changes of 0.045 g/L CCL micelles (1.0 μM NUMA
residues) (pH 7, 25 ꢀC) in the presence of 5.0 equiv of Naþ, Kþ, Agþ,
Co2þ, Pb2þ, Cu2þ, Zn2þ, Cd2þ, Fe2þ, Fe3þ, and Hg2þ, respectively.
Figure 5. (a) Fluorescence emission spectra and (b) fluorescence
intensity ratio changes (I468/I523) recorded for 0.045 g/L aqueous
solution of CCL micelles (1.0 μM NUMA residues) upon gradual
addition of Hg2þ (0-1.5 equiv) at 25 ꢀC. The spectra were obtained
20 min after Hg2þ addition (pH 7; λex = 395 nm; slit widths: Ex = 5 nm,
Em = 5 nm).
The spectra were obtained 20 min after addition of various ions (λex
=
390 nm, slit widths: Ex = 5 nm, Em = 5 nm).
Figure 6. Variation of relative fluorescence intensity recorded for 0.045 g/L
aqueous dispersion (1.0 μM NUMA residues) of (a) PEO113-b-P-
(NIPAM0.84-co-NAS0.16-co-NUMA)110 at 25 ꢀC (λem = 485 nm), (b)
CCL micelles at 40 ꢀC (λem = 468 nm), and (c) CCL micelles at 25 ꢀC
(λem = 468 nm) upon gradual addition of Hg2þ (0-1.5 equiv),
respectively (λex = 395 nm; slit widths: Ex = 5 nm, Em = 5 nm).
Figure 8. Optical photographs recorded under visible light (top) and UV
(365 nm, bottom) for 2.0 g/L CCL micelles 1 h after the addition of 5.0 equiv
of Naþ, Kþ, Pb2þ, Cu2þ, Hg2þ, Agþ, Zn2þ, Fe2þ, and Fe3þ, respectively.
CCL micelles bearing Hg2þ-reactive NUMA moieties can be
employed for cell imaging of Hg2þ ions. Moreover, the bright
field image shown in Figure 9c revealed that cells are generally
viable throughout the culture process. The above results augur
well for the further development of multifunctional CCL
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Figure 9. Fluorescent images (340/380 nm excitation filter in combination with longpass 420 nm barrier filter) of (a) HeLa cells incubated with0.45 g/L
CCL micelles (10.0 μM NUMA residues) at 37 ꢀC for 4 h, (b) HeLa cells incubated with 0.45 g/L CCL micelles (10.0 μM NUMA residues) at 37 ꢀC
for 4 h and then with 50.0 μM Hg2þ for 30 min, and (c) a bright field image of the cells in panel (b).
micelles capable of sensing, imaging, and controlled delivery
and release of therapeutic agents.
Universities, and Specialized Research Fund for the Doctoral
Program of Higher Education (SRFDP) is gratefully acknowledged.
’ CONCLUSION
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