Responsive nanogel-based dual fluorescent sensors for temperature and Hg2+ ions with enhanced detection sensitivity

Article abstract of DOI:10.1039/c0jm01828g

We report on the fabrication of thermoresponsive poly(N- isopropylacrylamide) nanogel-based dual fluorescent sensors for temperature and Hg²⁺ ions, and the effects of thermo-induced nanogel collapse on the detection sensitivity of Hg²⁺ ions. Near-monodisperse thermoresponsive nanogels were prepared via emulsion polymerization of N-isopropylacrylamide (NIPAM) and a novel 1,8-naphthalimide-based polarity-sensitive and Hg²⁺-reactive fluorescent monomer (NPTUA, 3). At room temperature, PNIPAM nanogels labeled with a single type of naphthalimide-based dye (NPTUA) can act as ratiometric Hg²⁺ probes at the nanomolar level. Upon heating above the phase transition temperature, the fluorescence intensity of NPTUA-labeled nanogels in the absence of Hg ²⁺ exhibit ～3.4-fold increase due to that NPTUA moieties are now located in a more hydrophobic microenvironment. Moreover, it was observed that the detection sensitivity to Hg²⁺ can be further improved above the nanogel phase transition temperature. At a nanogel concentration of 0.05 g L^-1 and in the same Hg²⁺ concentration range (0-3.0 equiv.), ～10 fold and ～57 fold increase in fluorescence emission intensity ratio changes can be achieved at 25 and 40 °C, respectively.

Full text of DOI:10.1039/c0jm01828g

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PAPER
www.rsc.org/materials | Journal of Materials Chemistry
Responsive nanogel-based dual fluorescent sensors for temperature and Hg2+
ions with enhanced detection sensitivity†
*
Changhua Li and Shiyong Liu
Received 9th June 2010, Accepted 4th August 2010
DOI: 10.1039/c0jm01828g
We report on the fabrication of thermoresponsive poly(N-isopropylacrylamide) nanogel-based dual
fluorescent sensors for temperature and Hg2+ ions, and the effects of thermo-induced nanogel collapse
on the detection sensitivity of Hg2+ ions. Near-monodisperse thermoresponsive nanogels were prepared
via emulsion polymerization of N-isopropylacrylamide (NIPAM) and a novel 1,8-naphthalimide-based
polarity-sensitive and Hg2+-reactive fluorescent monomer (NPTUA, 3). At room temperature,
PNIPAM nanogels labeled with a single type of naphthalimide-based dye (NPTUA) can act as
ratiometric Hg2+ probes at the nanomolar level. Upon heating above the phase transition temperature,
the fluorescence intensity of NPTUA-labeled nanogels in the absence of Hg2+ exhibit ꢀ3.4-fold increase
due to that NPTUA moieties are now located in a more hydrophobic microenvironment. Moreover, it
was observed that the detection sensitivity to Hg2+ can be further improved above the nanogel phase
transition temperature. At a nanogel concentration of 0.05 g Lꢁ1 and in the same Hg2+ concentration
range (0–3.0 equiv.), ꢀ10 fold and ꢀ57 fold increase in fluorescence emission intensity ratio changes can
ꢂ
be achieved at 25 and 40 C, respectively.
reaction of initially nonfluorescent rhodamine in the spirolactam
1. Introduction
form. However, the rhodamine-based small molecule Hg2+ che-
mosensors are subject to limitations such as poor water solubility
and considerable interferences from Ag+ and Cu2+ ions.45,59
Another approach for designing ratiometric metal ion sensors
relies on the utilization of intramolecular charge transfer (ICT)
processes by employing ICT-based fluorophores whose fluores-
cence emission band can shift remarkably upon binding or
reacting with target metal ions.46,48,60–62 A typical example is 4-
amino-1,8-naphthalimide derivatives. ICT-induced blue shift in
the emission peak will occur if the electron-donating capability of
amine moieties weakened by replacing with a less electron-rich or
electron-deficient substituent.60,63 Recently, Liu and Tian46
reported a novel small molecule ratiometric Hg2+ sensor based on
the 4-amino-1,8-naphthalimide thiourea derivative. The presence
of Hg2+ ions can induce a remarkable blue shift in the fluores-
cence emission. Compared to Hg2+ chemosensors based on leuco-
rhodamine derivatives, 1,8-naphthalimide-based chemosensors
possess additional advantages that the interference from Ag+ and
Cu2+ ions can be effectively eliminated. However, the reported
1,8-naphthalimide-based Hg2+ chemosensor is barely water-
soluble and this limits their practical applications.
In the past decades, the field of probing the presence and concen-
tration of heavy metal ions has received ever-increasing attention
owing to emerging environmental and human health issues.1–3 In
particular, as one of the most harmful metal ions, mercury can
cause considerable damage to human beings even at low concen-
trations due to bioaccumulation, long residence, and permanent
deterioration in the central nervous and endocrine systems.4 Thus,
the highlysensitive and selectivedetectionandimaging of Hg2+ ions
in tissues and organisms are quite crucial. In recent years, a variety
of fluorescent and colorimetric Hg2+ chemosensors have been
developed based on small molecules,5–23 conjugated polymers,24–26
nanoparticles,27–35 and biomolecules.27–30,36–42 Current efforts in this
field have focused on the invention of ratiometric,22,43–47 water-
soluble,25,44,48–51 and cell-permeable Hg2+-sensing ensembles.15,45,52
Recently, the concept of fluorescence resonance energy trans-
fer (FRET) has been introduced for designing chemosensors for
metal ions to achieve quantitative ratiometric sensing, which can
effectively eliminate background interferences and fluctuation of
detection conditions through the self-calibration of two emission
peaks.45,47,53–57 Based on the work of Tae and co-workers58 in
2005, Qian and co-workers45 reported the first example of a novel
small molecule FRET-based ratiometric Hg2+ sensor via the
covalent conjugation of boron dipyrromethene dye (BODIPY)
(FRET donor) with leuco-rhodamine derivative (FRET
acceptor), taking advantage of the Hg2+-triggered ring-opening
It has been well-established that thermoresponsive poly-
(N-isopropylacrylamide) (PNIPAM) nanogels exhibit thermo-
induced swelling/collapse behavior in aqueous dispersion, i.e.,
thermo-induced volume phase transitions (VPTs).64–67 Therefore,
we speculate that the incorporation of ICT-based metal ion-
sensing moieties into PNIPAM nanogels should enable the
development of highly sensitive and selective chemosensors with
combined advantages such as water solubility, biocompatibility,
and multifunctional integration. Most importantly, hydrophobic
domains within thermo-induced collapsed nanogels can help
achieve signal amplification of fluorescent reporter moieties in
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. E-mail: sliu@ustc.edu.cn
† Electronic supplementary information (ESI) available: Scheme S1 and
Fig. S1–S3. See DOI: 10.1039/c0jm01828g
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use. Acryloyl chloride (Sinopharm Chemical Reagent Co.) was
distilled prior to use. 4-Bromo-1,8-naphthalic anhydride (99%
purity), sodium dodecyl sulfate (SDS), n-propylamine, 1,2-
ethylenediamine, and all other reagents were purchased from
Sinopharm Chemical Reagent Co., Ltd., and used as received.
Acetonitrile (MeCN) was dried over CaH2 and distilled just prior
to use. Toluene and 1,4-dioxane were distilled over sodium
shavings and benzophenone just prior to use. Nitrate salts (Hg2+,
Na+, K+, Ag+, Ca2+, Cu2+, Zn2+, Cd2+, Pb2+, Fe2+, Co2+, Ni2+ and
Mn2+) were used for all sensing experiments. Water was deion-
ized with a Milli-Q SP reagent water system (Millipore) to
a specific resistivity of 18.4 MU cm. Acryloyl isothiocyanate was
available from our previous work.59
Sample synthesis
Synthetic schemes employed for the preparation of 1,8-
naphthalimide-based polarity-sensitive and Hg2+-reactive
monomer (NPTUA, 3), are shown in Scheme S1.†
Preparation of 4-bromo-N-n-propyl-1,8-naphthalimide (NP–Br, 1).
A 500 mL round-bottom flask was charged with 4-bromo-1,8-
naphthalic anhydride (13.85 g, 50 mmol), n-propylamine (2.96 g,
50 mmol), and 200 mL 1,4-dioxane. The reaction mixture was
stirred at reflux for 8 h. After cooling to room temperature, the
suspension was poured into 800 mL ice water and then filtrated.
After drying in a vacuum oven overnight at room temperature,
1 was obtained as a slightly gray solid (14.23 g, yield: 89%).
1H NMR (CDCl3, d, ppm, TMS, Fig. S1a†): 8.66 (1H, 5-CH), 8.57
(1H, 7-CH), 8.41 (1H, 2-CH), 8.04 (1H, 3-CH), 7.85 (1H,
6-CH), 4.14 (2H, -CH2CH2CH3), 1.84–1.69 (2H, -CH2CH2CH3),
and 1.07–0.95 (3H, -CH2CH2CH3).
Scheme 1 A schematic illustration of the construction of thermores-
ponsive PNIPAM nanogel-based dual fluorescent sensors for tempera-
ture and Hg2+ ions with enhanced detection sensitivity.
sensing ensembles,59,68–71 which can further enhance the detection
sensitivity of metal ions (Scheme 1).
In the current work, we report on the synthesis of
thermoresponsive PNIPAM nanogels copolymerized with 1,8-
naphthalimide-based polarity-sensitive and Hg2+-reactive
monomer (NPTUA, 3), which can serve as dual fluorescent
sensors for Hg2+ ions and temperature (Schemes 1 and S1†).
Near-monodisperse thermoresponsive P(NIPAM-co-NPTUA)
nanogels were prepared via emulsion copolymerization of N-
isopropylacrylamide (NIPAM) and NPTUA (3) in the presence
of N,N0-methylenebis(acrylamide) (BIS) and an anionic surfac-
tant. The detection sensitivity and selectivity of as-synthesized
nanogels to Hg2+ ions at varying temperatures (below and above
the nanogel VPT temperatures) have been investigated in detail.
We found that NPTUA-labeled PNIPAM nanogels in their
swollen state can selectively react with Hg2+ over other metal
ions, leading to prominent blue shift in fluorescence emission
peak. Thus, PNIPAM nanogels labeled with a single type of
naphthalimide-based dye can act as ratiometric Hg2+ probes at
the nanomolar level. Moreover, Hg2+ detection sensitivity of
NPTUA-labeled PNIPAM nanogels can be considerably
enhanced via thermo-induced nanogel collapse at elevated
temperatures (Scheme 1).
Preparation of 4-(2-aminoethyl)amino-N-n-propyl-1,8-naph-
thalimide (NP-NH2, 2). NP-NH2 was prepared by refluxing
1 with an excess of 1,2-ethylenediamine. Into a 100 mL round-
bottom flask equipped with a magnetic stirring bar and reflux
condenser, 1 (3.18 g, 10 mmol) and 1,2-ethylenediamine (50 mL)
were charged. The reaction mixture was stirred at reflux for 10 h.
After removing all the solvents on a rotary evaporator, the
residues were dissolved in THF and precipitated into an excess of
water. The crude compound was further purified by recrystalli-
zation from toluene. After drying in a vacuum oven overnight at
room temperature, NP-NH2 (2) was obtained as an orange
crystal (2.32 g, yield: 78%). 1H NMR (d6-DMSO, d, ppm, TMS,
Fig. S1b†): 8.68 (1H, 5-CH), 8.41 (1H, 7-CH), 8.24 (1H, 2-CH),
7.66 (1H, 6-CH), 6.79 (1H, 3-CH), 3.96 (2H, -CH2CH2CH3), 2.86
(2H, -CH2NH2) 1.68–1.54 (2H, -CH2CH2CH3), and 0.99–0.85
(3H, -CH2CH2CH3).
Preparation of 1,8-naphthalimide-based monomer (NPTUA, 3).
NP-NH2 (2, 0.59 g, 2.0 mmol) was dissolved in 20 mL of dry
MeCN. Freshly prepared acryloyl isothiocyanate (0.23 g,
2.0 mmol) in dry MeCN (10 mL) was then added dropwise over
2. Experimental section
Materials
ꢂ
N-Isopropylacrylamide (NIPAM, 97%, Tokyo Kasei Kagyo
Co.) was purified by recrystallization from a mixture of benzene
and n-hexane (1/3, v/v). Potassium persulfate (KPS) and N,N0-
methylenebis(acrylamide) (BIS) were recrystallized from meth-
anol and ethanol, respectively, and then stored at ꢁ20 ꢂC prior to
0.5 h. The reaction mixture was stirred at 40 C for 3 h. After
cooling to room temperature, the precipitates were collected by
filtration and washed with dry MeCN for three times. The crude
product was further purified by recrystallization from MeCN.
After drying in a vacuum oven overnight at room temperature,
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NPTUA was obtained as an orange powder (0.55 g, yield: 68%).
1H NMR (d6-DMSO, d, ppm, TMS, Fig. S1c†): 8.67 (1H, 5-CH),
8.44 (1H, 7-CH), 8.25 (1H, 2-CH), 7.69 (1H, 6-CH), 6.98 (1H, 3-
CH), 6.56 (1H, -CH ¼ CHH), 6.36 (1H, -CH ¼ CHH), 5.94
(1H, -CH ¼ CHH), 3.98 (4H, -CH2CH2CH3, Ar-NHCH2-), 3.67
(2H, -CH2NHC(¼S)-) 1.72–1.51 (2H, -CH2CH2CH3), and 0.96–
0.82 (3H, -CH2CH2CH3).
Atomic force microscope (AFM). AFM measurements were
performed on a Digital Instrument Multimode Nanoscope IIID
operating in the tapping mode under ambient conditions. Silicon
cantilever (RFESP) with resonance frequency of ꢀ80 kHz and
spring constant of ꢀ3 N mꢁ1 was used. The set-point amplitude
ratio was maintained at 0.7 to minimize sample deformation
induced by the tip. The sample was prepared by dip-coating
0.01 g Lꢁ1 nanogel dispersion onto freshly cleaved mica surface
and followed by natural drying.
Synthesis of NPTUA-labeled thermoresponsive P(NIPAM-co-
NPTUA) nanogels. Typical procedures employed for the prepa-
ration of PNIPAM nanogels labeled with NPTUA moieties are
as follows. Into a three-neck round-bottom flask equipped with
a mechanical Teflon stirrer, a reflux condenser, and a nitrogen-
bubbling tube, NIPAM (0.5 g, 4.32 mmol), BIS (15 mg, 97.29
mmol), SDS (4 mg), and deionized water (50 mL) were charged.
After degassing by bubbling with N2 for 30 min and thermo-
Fluorescence measurements. Fluorescence spectra were recor-
ded 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 both excitation and emission. For all nanogel-based Hg2+
fluorescent sensing experiments, nanogel concentrations are fixed
at 0.05 g Lꢁ1 ([NPTUA] ¼ 5.0 ꢃ 10ꢁ7 M).
ꢂ
stating at 70 C under mechanical stirring (400 rpm), KPS (25
mg, 92.48 mmol) was introduced to initiate the emulsion poly-
merization. NPTUA monomer (3 mg, 7.31 mmol) in a mixture of
DMSO (0.4 mL) and acetone (5 mL) was then added dropwise
over ꢀ4 min. The polymerization was conducted under stirring
for 7 h. After cooling to room temperature, the dispersion was
passed through glass wool in order to remove particulates and
then purified by dialysis (cellulose membrane, molecular weight
cut-off: 14 kDa) against deionized water for 5 days. Fresh
deionized water was replaced approximately every 4 h.
3. Results and discussion
Synthesis of P(NIPAM-co-NPTUA) nanogels
Synthetic routes employed for the preparation of novel 1,8-
naphthalimide-based polarity-sensitive and Hg2+-reactive
fluorescent monomer (NPTUA, 3) and NPTUA-labeled ther-
moresponsive nanogels are shown in Scheme S1. The reaction of
4-bromo-1,8-naphthalic anhydride with n-propylamine leads to
4-bromo-N-n-propyl-1,8-naphthalimide (NP-Br, 1) with a yield
of 89%. Its chemical structure was confirmed by 1H NMR
analysis (Fig. S1†). This was followed by the nucleophilic
substitution reaction of NP-Br with an excess of 1,2-ethylenedi-
amine. In the final step, the target 1,8-naphthalimide-based
monomer (NPTUA, 3) was obtained by the reaction of NP-NH2
with acryloyl isothiocyanate (Scheme S1†). The chemical struc-
tures of both NP-NH2 and NPTUA (3) were confirmed by
1H NMR analysis (see Fig. S1†).
Characterization
Nuclear magnetic resonance (NMR) spectroscopy. All 1H NMR
spectra were recorded on a Bruker AV400 NMR spectrometer
(resonance frequency of 400 MHz for 1H) operated in the Fourier
transform mode. CDCl3 and d6-DMSO were used as the
solvents.
UV-vis measurements. All UV-Vis spectra were acquired on
P(NIPAM-co-NPTUA) nanogels were synthesized via free
radical emulsion copolymerization of NIPAM and NPTUA in
a Unico UV/Vis 2802PCS spectrophotometer.
ꢂ
the presence of BIS and SDS at around neutral pH and 70 C.
Laser light scattering (LLS). A commercial spectrometer
(ALV/DLS/SLS-5022F) equipped with a multi-tau digital time
correlator (ALV5000) and a cylindrical 22 mW UNIPHASE He–
Ne laser (l0 ¼ 632 nm) as the light source was employed for
dynamic laser light scattering (LLS) measurements. Scattered
light was collected at a fixed angle of 90ꢂ 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.
As-synthesized nanogels with a designed cross-linking density of
Stopped-flow fluorescence measurements. Stopped-flow fluo-
rescence studies were carried out using a Bio-Logic SFM300/S
stopped-flow instrument. The SFM-3/S is a three-syringe (10
mL) instrument in which all step-motor-driven syringes (S1, S2,
and S3) can be operated independently to carry out single- or
double-mixing. The SFM-300/S stopped-flow device is attached
to the MOS-250 spectrometer. Kinetic data were fitted using the
program Biokine (Bio-Logic). For the fluorescence emission
detection, the excitation and emission wavelengths (10 nm slit
widths in both cases) were set at 390 and 482 nm, respectively.
The typical dead time is 2.6 ms using the FC-15 flow cell.
Fig. 1 Temperature-dependence of the intensity average hydrodynamic
radius, <Rh>, obtained for 0.05 g Lꢁ1 aqueous dispersion of P(NIPAM-
co-NPTUA) nanogels ([NPTUA] ¼ 5.0 ꢃ 10ꢁ7 M). The inset shows
typical hydrodynamic radius distributions, f(Rh), of P(NIPAM-co-
NPTUA) nanogels at 25 and 40 ꢂC, respectively.
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Fig. 3 Absorption spectra recorded for 0.5 g Lꢁ1 aqueous dispersion of
P(NIPAM-co- NPTUA) nanogels ([NPTUA] ¼ 5.0 ꢃ 10ꢁ6 M) upon
gradual addition of Hg2+ (0–2.5 equiv.) at 25 ꢂC. Each spectrum was
acquired 10 min after Hg2+ addition.
derivatives can affect both the ICT process and the fluorescence
emission bands.46,48,63 If the amine moieties rendered less
electron-rich, ICT-induced blue shifts in the emission maxima
can be achieved. In the current work, we designed a novel poly-
merizable 1,8-naphthalimide-based monomer with the 4-position
modified with a substituent that could be selectively uncaged by
Hg2+ ions, and this process leads to the transformation of
secondary amine to tertiary amine residues (Scheme 1).
Considering its poor water-solubility, we copolymerized
NPTUA monomers into thermoresponsive PNIPAM nanogels
to solve this limitation. In the absence of Hg2+ ions, P(NIPAM-
co-NPTUA) nanogels display one major absorption band
centered at 438 nm with a corresponding green-colored fluores-
cence emission maximum at 528 nm (Fig. 3 and 4). The addition
of Hg2+ ions in aqueous media transforms the thiourea moiety
within NPTUA into the imidazoline moiety with considerably
weakened electron-donating ability,46,48 which is accompanied
with significant reduction in electron delocalization as compared
to that occurred in the NPTUA precursor (Scheme 1). The Hg2+-
triggered intramolecular cyclization of thiourea moieties results
in ICT-induced blue shifts in fluorescence emissions. Typical
absorption and fluorescence emission spectra recorded for the
aqueous dispersion of NPTUA-labeled nanogels upon gradual
addition of Hg2+ ions are shown in Fig. 3 and 4.
Fig. 2 (a) SEM and (b) AFM height images (Z range is 50 nm) recorded
for aqueous dispersion of P(NIPAM-co-NPTUA) nanogels at 25 ꢂC.
3.0 wt% were characterized by LLS, SEM, and AFM analysis
(Fig. 1 and 2). Aqueous dispersion of P(NIPAM-co-NPTUA)
nanogels exhibit an intensity-average hydrodynamic radius,
<Rh>, of 125 nm and a polydispersity index, m2/G2, of 0.012 at
25 ꢂC, as revealed by dynamic LLS (Fig. 1). Upon heating to
40 ꢂC, which is above the volume phase transition (VPT)
temperature of conventional PNIPAM nanogels, initially swollen
nanogels collapse and dynamic LLS reveals an <Rh> of 57 nm
and a polydispersity of 0.018. Thus, heating the nanogel
ꢂ
dispersion from 25 to 40 C led to ꢀ10.5 times shrinkage in the
hydrodynamic volumes. The molar concentration of NPTUA
residues in 10.0 g Lꢁ1 nanogel dispersion was determined to be
ꢀ1.0 ꢃ 10ꢁ4 M based on UV absorbance calibration curve.
SEM and AFM images obtained by drying the aqueous
dispersion of P(NIPAM-co-NPTUA) nanogels at 25 ꢂC revealed
the presence of fairly monodisperse and spherical nanoparticles
with average sizes of ꢀ125 and 110 nm, respectively. This agrees
reasonably well with that determined by dynamic LLS for
collapsed nanogels (Fig. 1). It is well-known that SEM and AFM
determine nanoparticle dimensions in the dry state, whereas
dynamic LLS report the intensity-average dimensions in the
solution.
As shown in Fig. 3, the UV-vis absorbance intensity of the
nanogel dispersion at 438 nm considerably decreases upon
gradual addition of Hg2+ ions (0–2.5 equiv. relative to that of
NPTUA residues), accompanied with the appearance and
growth of a new absorption band at ꢀ350 nm. In the Hg2+
concentration range investigated, the absorbance intensity
exhibits approximately 3.2-fold enhancement at 350 nm and
ꢀ5.1-fold decrease at 438 nm. This can be clearly ascribed to
Hg2+-induced formation of imidazoline moieties within NPTUA
(Scheme 1). Similar processes have been proposed for small
molecule naphthalimide-based derivatives possessing compa-
rable chemical structures.46,48
P(NIPAM-co-NPTUA) nanogels as dual sensors for Hg2+ ions
and temperature
The aqueous dispersion of P(NIPAM-co-NPTUA) nanogels
(0.05 g Lꢁ1, [NPTUA] ¼ 5.0 ꢃ 10ꢁ7 M) in the absence of Hg2+
ions exhibit relatively intense fluorescence emission at 528 nm
when excited at 390 nm.46 This indicates that the incorporation of
NPTUA moieties into thermoresponsive PNIPAM nanogels
does not alter their photophysical properties. As shown in
We then investigated the Hg2+-sensing capability of NPTUA-
labeled PNIPAM nanogels in their swollen state at 25 ꢂC. It has
been well-documented that the modulation of electron-donating
capabilities of amine substituent on 1,8-naphthalimide-based
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Fig. 5 (a) Fluorescence emission spectra and (b) fluorescence intensity
ratio changes (I482/I528; lex ¼ 390 nm, slit widths: Ex. 5 nm, Em. 5 nm)
recorded for 0.05 g Lꢁ1 aqueous dispersion of P(NIPAM-co-NPTUA)
nanogels ([NPTUA] ¼ 5.0 ꢃ 10ꢁ7 M, 25 ꢂC) within 10 min after the
Fig. 4 (a) Fluorescence emission spectra and (b) fluorescence intensity
ratio changes (I482/I528; lex ¼ 390 nm, slit widths: Ex. 5 nm, Em. 5 nm)
recorded for 0.05 g Lꢁ1 aqueous dispersion of P(NIPAM-co-NPTUA)
nanogels ([NPTUA] ¼ 5.0 ꢃ 10ꢁ7 M) upon gradual addition of Hg2+ (0–3
equiv.) at 25 ꢂC. Each spectrum was acquired 10 min after Hg2+ addition.
The inset in (b) shows the determination of the detection limit.
addition of 5 equiv. of Hg2+, Na+, K+, Ag+, Ca2+, Cu2+, Zn2+, Cd2+, Pb2+
Fe2+, Co2+, Ni2+, and Mn2+, respectively.
,
similar changes in the absorption and fluorescence emission
spectra, but it takes ꢀ18 h to complete. This is again in agree-
ment with that reported for small molecule naphthalimide urea
derivatives.46,48 By employing the stopped-flow technique, we
further observed that the Hg2+-induced fluorescence ‘‘blue shift’’
process goes to completion within ꢀ6 min (Fig. S2†), which is
very fast compared to that of Ag+ ions. Thus, we can facilely
eliminate the interference of Ag+ by employing suitable detection
time windows. Based on the above results, we conclude that
P(NIPAM-co-NPTUA) nanogels possess a high specificity and
selectivity for Hg2+ ions on a ratiometric basis.
Fig. 4a, upon gradual addition of Hg2+ ions, a new fluorescence
emission band at 482 nm substantially increases and then stabi-
lizes above ꢀ2.0 equiv. of Hg2+ ions (relative to NPTUA resi-
dues), which is accompanied with the considerable decrease in
fluorescence emission intensity at 528 nm. The dramatic blue
shift of fluorescence emission feature is also clearly evident from
fluorescence intensity ratio changes, I482/I528, upon Hg2+ addition
(Fig. 4b). The emission intensity ratios (I482/I528) of imidazoline-
to thiourea-derivatized naphthalimide upon excitation at 390 nm
vary from 0.20 in the absence of Hg2+ to ꢀ1.98 after complete
Hg2+-induced conversion to imidazoline-derivatized naph-
thalimide (in the presence of 3.0 equiv. Hg2+ ions), i.e., a ca. 10-
fold change in emission intensity ratios. If we define the detection
limit as the Hg2+ concentration at which a 10% change in fluo-
rescence intensity ratio relative to the blank sample can be
measured by employing 0.05 g Lꢁ1 aqueous dispersion of
P(NIPAM-co-NPTUA) nanogels, the Hg2+ detection limit is
determined to be ꢀ5.1 nM (Fig. 4b).
It has been well-established that hydrophobic microenviron-
ments can typically enhance the quantum yields of fluorescent
dyes.67–70 Our recent research interest involves the modulation of
emission of caged fluorescent dyes by integrating them into
stimuli-responsive polymeric assemblies and nanoparticles to
achieve multifunctionality, water-solubility, and biocompati-
bility, aiming at developing novel drug nanocarriers with
imaging and sensing capabilities.59,72 As P(NIPAM-co-NPTUA)
nanogels collapse above the phase transition temperature (Fig. 1)
accompanied with the formation of more hydrophobic micro-
environment, we speculate that the incorporation of NPTUA
moieties into thermoresponsive nanogels can act as a novel
fluorescent thermometer. We then investigated the temperature-
dependent fluorescence emission of NPTUA-labeled nanogel
dispersions in the absence of Hg2+ ions. As shown in Fig. 7,
heating the nanogel dispersion from 25 to 40 ꢂC apparently leads
to the increase of emission intensity at 528 nm. Concomitantly,
a slight blue shift of the emission maximum from 528 to 511 nm
can be observed. This indicated that the quantum yield of
NPTUA dyes can be considerably enhanced when the nanogel
The selectivity of P(NIPAM-co-NPTUA) nanogels for Hg2+
ion sensing was then investigated (Fig. 5 and 6). Among a series
of cations including Na+, K+, Ag+, Ca2+, Cu2+, Zn2+, Cd2+, Pb2+,
Fe2+, Co2+, Ni2+, Mn2+, and Hg2+ (5 equiv.), only Hg2+ exhibits
the apparent blue shift of UV-vis absorption (yellow to colorless)
and fluorescence emission (green to blue transition) within 10
min upon addition. The Hg2+-induced absorption and fluores-
cence emission changes can also be visualized by the naked eye
(Fig. 6). The above results indicate that the incorporation of
NPTUA moieties into PNIPAM nanogels does not alter their
selective Hg2+-sensing capability. Ag+ ions can also induce
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Fig. 6 Optical photographs recorded under visible light (top) and UV lamp (365 nm, bottom) for 1.0 g Lꢁ1 aqueous dispersion of P(NIPAM-co-
NPTUA) nanogels ([NPTUA] ¼ 1.0 ꢃ 10ꢁ5 M) within 10 min after the addition of 5 equiv. of Hg2+, Na+, K+, Ag+, Ca2+, Cu2+, Zn2+, Cd2+, Pb2+, Fe2+
,
Co2+, Ni2+, and Mn2+, respectively.
Effects of thermo-induced nanogel collapse on Hg2+-sensing
Thermo-induced enhancement of fluorescence emission of
P(NIPAM-co-NPTUA) nanogel dispersions can be further
utilized as an approach to achieve signal amplification in Hg2+-
sensing experiments. Fig. 8a shows the fluorescence emission
Fig. 7 (a) Fluorescence emission spectra and (b) fluorescence intensity
changes (lex ¼ 390 nm, lem ¼ 511 nm; slit widths: Ex. 5 nm, Em. 5 nm)
recorded for 0.05 g Lꢁ1 aqueous dispersion of P(NIPAM-co-NPTUA)
nanogels ([NPTUA] ¼ 5.0 ꢃ 10ꢁ7 M) in the temperature range 25–42 ꢂC.
matrix gets more hydrophobic due to thermo-induced collapse.
ꢂ
Fig. 8 (a) Fluorescence emission spectra and (b) fluorescence intensity
ratio changes (I457/I511; lex ¼ 390 nm, slit widths: Ex. 5 nm, Em. 5 nm)
recorded at 40 ꢂC for 0.05 g Lꢁ1 aqueous dispersion of P(NIPAM-co-
NPTUA) nanogels ([NPTUA] ¼ 5.0 ꢃ 10ꢁ7 M) upon gradual addition of
Hg2+ (0–3 equiv.). Each spectrum was acquired 10 min after Hg2+ addi-
tion. The inset in (b) shows the determination of Hg2+ detection limit.
In the narrow temperature range of 30–38 C, the fluorescence
emission intensity at 528 nm exhibits ꢀ2.8 times increase
(Fig. 7b). Thus, NPTUA-labeled thermoresponsive nanogels can
also act as fluorescent thermometers in the absence of mercury
ions.
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spectra recorded for the aqueous dispersion of P(NIPAM-co-
NPTUA) nanogels upon gradual addition of Hg2+ ions at 40 ꢂC.
We can clearly observe the appearance and substantial increase
of a new fluorescence emission band at 457 nm upon addition of
Hg2+ ions, accompanied with the considerable decrease of emis-
sion intensity at 511 nm. The new emission band at ꢀ457 nm
tends to stabilize out in the presence of >2.0 equiv. of Hg2+ ions
(relative to NPTUA residues). Fig. 8b plots the Hg2+ concentra-
of our knowledge, this work represents the first report of
successful integration of stimuli-responsive nanogels with well-
developed small molecule reaction-based selective metal ion
sensing moieties.
Acknowledgements
The financial support of National Natural Scientific Foundation
of China (NNSFC) Project (20874092) and Specialized Research
Fund for the Doctoral Program of Higher Education (SRFDP)
are gratefully acknowledged.
tion-dependence of fluorescence intensity ratio changes, I457/I511
,
which is again ascribed to Hg2+-induced transformation of
naphthalimide-thiourea to naphthalimide-imidazoline deriva-
tives. We can tell from Fig. 8b that the emission intensity ratios,
I457/I511, increase from 0.04 for the blank sample to 2.29 in the
presence of 3.0 equiv. of Hg2+ ions.
References
In the same Hg2+ concentration range (0–3.0 equiv.) for 0.05 g
Lꢁ1 P(NIPAM-co-NPTUA) nanogel dispersion, ꢀ10 fold and
ꢀ57 fold increase in fluorescence emission intensity ratio changes
1 M. Beija, C. A. M. Afonso and J. M. G. Martinho, Chem. Soc. Rev.,
2009, 38, 2410–2433.
2 J. S. Kim and D. T. Quang, Chem. Rev., 2007, 107, 3780–3799.
3 L. Basabe-Desmonts, D. N. Reinhoudt and M. Crego-Calama, Chem.
Soc. Rev., 2007, 36, 993–1017.
ꢂ
can be achieved at 25 and 40 C, respectively. This means that
Hg2+ detection above the VPT temperature of P(NIPAM-co-
NPTUA) nanogels will be more sensitive, as compared to that
below the VPT temperatures. We thus established thermo-
induced collapse of thermoresponsive PNIPAM nanogels can be
successfully utilized to achieve signal amplification and much
better detection sensitivity.
From Fig. 8b, we can also read out a Hg2+ detection limit of
2.5 nM for 0.05 g Lꢁ1 P(NIPAM-co-NPTUA) nanogel disper-
sions at 40 ꢂC, which is again much better than that at 25 ꢂC
(5.1 nM). Fig. S3† plots the Hg2+ ion concentration dependence
of normalized fluorescence intensities of emission band ascribed
4 T. W. Clarkson, L. Magos and G. J. Myers, N. Engl. J. Med., 2003,
349, 1731–1737.
5 D. G. Cho and J. L. Sessler, Chem. Soc. Rev., 2009, 38, 1647–1662.
6 E. M. Nolan and S. J. Lippard, Chem. Rev., 2008, 108, 3443–3480.
7 H. N. Kim, M. H. Lee, H. J. Kim, J. S. Kim and J. Yoon, Chem. Soc.
Rev., 2008, 37, 1465–1472.
8 E. M. Nolan and S. J. Lippard, J. Am. Chem. Soc., 2007, 129, 5910–
5918.
9 C. S. Lim, D. W. Kang, Y. S. Tian, J. H. Han, H. L. Hwang and
B. R. Cho, Chem. Commun., 2010, 46, 2388–2390.
10 D. Wu, A. B. Descalzo, F. Weik, F. Emmerling, Z. Shen, X. Z. You
and K. Rurack, Angew. Chem., Int. Ed., 2008, 47, 193–197.
11 S. V. Wegner, A. Okesli, P. Chen and C. A. He, J. Am. Chem. Soc.,
2007, 129, 3474–3475.
12 J. V. Ros-Lis, M. D. Marcos, R. Martinez-Manez, K. Rurack and
J. Soto, Angew. Chem., Int. Ed., 2005, 44, 4405–4407.
13 A. Ono and H. Togashi, Angew. Chem., Int. Ed., 2004, 43, 4300–4302.
14 E. M. Nolan and S. J. Lippard, J. Am. Chem. Soc., 2003, 125, 14270–
14271.
15 W. Y. Lin, X. W. Cao, Y. D. Ding, L. Yuan and L. L. Long, Chem.
Commun., 2010, 46, 3529–3531.
16 X. F. Guo, X. H. Qian and L. H. Jia, J. Am. Chem. Soc., 2004, 126,
2272–2273.
17 J. J. Du, J. L. Fan, X. J. Peng, P. P. Sun, J. Y. Wang, H. L. Li and
S. G. Sun, Org. Lett., 2010, 12, 476–479.
18 A. B. Descalzo, R. Martinez-Manez, R. Radeglia, K. Rurack and
J. Soto, J. Am. Chem. Soc., 2003, 125, 3418–3419.
19 A. Coskun and E. U. Akkaya, J. Am. Chem. Soc., 2006, 128, 14474–
14475.
20 E. Coronado, J. R. Galan-Mascaros, C. Marti-Gastaldo,
E. Palomares, J. R. Durrant, R. Vilar, M. Gratzel and
M. K. Nazeeruddin, J. Am. Chem. Soc., 2005, 127, 12351–12356.
21 M. G. Choi, Y. H. Kim, J. E. Namgoong and S. K. Chang, Chem.
Commun., 2009, 3560–3562.
22 X. H. Cheng, Q. Q. Li, J. G. Qin and Z. Li, ACS Appl. Mater.
Interfaces, 2010, 2, 1066–1072.
23 A. Caballero, R. Martinez, V. Lloveras, I. Ratera, J. Vidal-Gancedo,
K. Wurst, A. Tarraga, P. Molina and J. Veciana, J. Am. Chem. Soc.,
2005, 127, 15666–15667.
to the naphthalimide-imidazoline derivative at 25 ꢂC (lem
¼
482 nm) and 40 ꢂC (lem ¼ 457 nm), respectively. In both cases, we
can clearly observe the almost linear increase of emission inten-
sities with increasing Hg2+ concentrations. Similarly, if we define
the detection limit as the Hg2+ concentration at which a 10%
emission enhancement can be measured by employing 0.05 g Lꢁ1
aqueous dispersion of the P(NIPAM-co-NPTUA) nanogels, the
Hg2+ detection limits again considerably improve from ꢀ13.3 nM
ꢂ
to ꢀ4.1 nM upon the increase in temperature from 25 to 40 C
(Fig. S3†).
4. Conclusions
In summary, we reported the fabrication of thermoresponsive
PNIPAM nanogel-based dual fluorescent sensors for tempera-
ture and Hg2+ ions, and investigated the effects of thermo-
induced nanogel collapse on the detection sensitivity. A novel
1,8-naphthalimide-based polarity-sensitive and Hg2+-reactive
fluorescent monomer (NPTUA) was synthesized. Near-
monodisperse thermoresponsive NPTUA-labeled PNIPAM
nanogels were prepared via the free radical emulsion copoly-
merization technique. The nanogel-based chemosensors possess
a high selectivity and sensitivity for Hg2+ at room temperature,
24 S. W. Thomas, G. D. Joly and T. M. Swager, Chem. Rev., 2007, 107,
1339–1386.
25 C. J. Qin, X. F. Wu, B. X. Gao, H. Tong and L. X. Wang,
Macromolecules, 2009, 42, 5427–5429.
26 X. F. Liu, Y. L. Tang, L. H. Wang, J. Zhang, S. P. Song, C. H. Fan
and S. Wang, Adv. Mater., 2007, 19, 1471–1474.
27 J. S. Lee, M. S. Han and C. A. Mirkin, Angew. Chem., Int. Ed., 2007,
46, 4093–4096.
28 W. W. Guo, J. P. Yuan and E. K. Wang, Chem. Commun., 2009,
3395–3397.
29 R. Freeman, T. Finder and I. Willner, Angew. Chem., Int. Ed., 2009,
48, 7818–7821.
30 J. S. Lee and C. A. Mirkin, Anal. Chem., 2008, 80, 6805–6808.
achieving
a detection limit at the nanomolar level on
a ratiometric basis. Thermo-induced collapse of P(NIPAM-co-
NPTUA) nanogel have been successfully utilized to further
enhance the detection sensitivity. In the same Hg2+ concentration
range (0–3.0 equiv.) for 0.05 g Lꢁ1 nanogel dispersion, ꢀ10 fold
and ꢀ57 fold increase in fluorescence emission intensity ratio
changes can be achieved at 25 and 40 ꢂC, respectively. To the best
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View Article Online
31 M. Rex, F. E. Hernandez and A. D. Campiglia, Anal. Chem., 2006, 78,
445–451.
52 S. Yoon, E. W. Miller, Q. He, P. H. Do and C. J. Chang, Angew.
Chem., Int. Ed., 2007, 46, 6658–6661.
32 S. J. Lee, J. E. Lee, J. Seo, I. Y. Jeong, S. S. Lee and J. H. Jung, Adv.
Funct. Mater., 2007, 17, 3441–3446.
33 C. C. Huang, Z. Yang, K. H. Lee and H. T. Chang, Angew. Chem.,
Int. Ed., 2007, 46, 6824–6828.
34 C. C. Huang and H. T. Chang, Anal. Chem., 2006, 78, 8332–8338.
35 G. K. Darbha, A. Ray and P. C. Ray, ACS Nano, 2007, 1, 208–214.
36 C. W. Liu, C. C. Huang and H. T. Chang, Anal. Chem., 2009, 81,
2383–2387.
53 Z. X. Han, X. B. Zhang, L. Zhuo, Y. J. Gong, X. Y. Wu, J. Zhen,
C. M. He, L. X. Jian, Z. Jing, G. L. Shen and R. Q. Yu, Anal.
Chem., 2010, 82, 3108–3113.
54 E. M. W. M. Van Dongen, L. M. Dekkers, K. Spijker, E. W. Meijer,
L. W. J. Klomp and M. Merkx, J. Am. Chem. Soc., 2006, 128, 10754–
10762.
55 W. Qiao, M. Mooney, A. J. Bird, D. R. Winge and D. J. Eide, Proc.
Natl. Acad. Sci. U. S. A., 2006, 103, 8674–8679.
37 X. J. Zhu, S. T. Fu, W. K. Wong, H. P. Guo and W. Y. Wong, Angew.
Chem., Int. Ed., 2006, 45, 3150–3154.
56 V. S. Jisha, A. J. Thomas and D. Ramaiah, J. Org. Chem., 2009, 74,
6667–6673.
38 L. B. Zhang, L. Tao, B. L. Li, L. Jing and E. K. Wang, Chem.
Commun., 2010, 46, 1476–1478.
39 S. J. Ou, Z. H. Lin, C. Y. Duan, H. T. Zhang and Z. P. Bai, Chem.
Commun., 2006, 4392–4394.
57 Z. G. Zhou, M. X. Yu, H. Yang, K. W. Huang, F. Y. Li, T. Yi and
C. H. Huang, Chem. Commun., 2008, 3387–3389.
58 Y. K. Yang, K. J. Yook and J. Tae, J. Am. Chem. Soc., 2005, 127,
16760–16761.
40 Y. Miyake, H. Togashi, M. Tashiro, H. Yamaguchi, S. Oda,
M. Kudo, Y. Tanaka, Y. Kondo, R. Sawa, T. Fujimoto,
T. Machinami and A. Ono, J. Am. Chem. Soc., 2006, 128, 2172–2173.
41 M. Matsushita, M. M. Meijler, P. Wirsching, R. A. Lerner and
K. D. Janda, Org. Lett., 2005, 7, 4943–4946.
59 J. M. Hu, C. H. Li and S. Y. Liu, Langmuir, 2010, 26, 724–729.
60 Z. C. Xu, Y. Xiao, X. H. Qian, J. N. Cui and D. W. Cui, Org. Lett.,
2005, 7, 889–892.
61 X. J. Peng, J. J. Du, J. L. Fan, J. Y. Wang, Y. K. Wu, J. Z. Zhao,
S. G. Sun and T. Xu, J. Am. Chem. Soc., 2007, 129, 1500.
62 Y. Shiraishi, S. Sumiya and T. Hirai, Org. Biomol. Chem., 2010, 8,
1310–1314.
42 I. B. Kim and U. H. F. Bunz, J. Am. Chem. Soc., 2006, 128, 2818–
2819.
43 C. Y. Li, X. B. Zhang, L. Qiao, Y. Zhao, C. M. He, S. Y. Huan,
L. M. Lu, L. X. Jian, G. L. Shen and R. Q. Yu, Anal. Chem., 2009,
81, 9993–10001.
44 M. Q. Tian and H. Ihmels, Chem. Commun., 2009, 3175–3177.
45 X. L. Zhang, Y. Xiao and X. H. Qian, Angew. Chem., Int. Ed., 2008,
47, 8025–8029.
46 B. Liu and H. Tian, Chem. Commun., 2005, 3156–3158.
47 A. Ben Othman, J. W. Lee, J. S. Wu, J. S. Kim, R. Abidi, P. Thuery,
J. M. Strub, A. Van Dorsselaer and J. Vicens, J. Org. Chem., 2007, 72,
7634–7640.
63 D. Srikun, E. W. Miller, D. W. Dornaille and C. J. Chang, J. Am.
Chem. Soc., 2008, 130, 4596–4597.
64 I. Berndt and W. Richtering, Macromolecules, 2003, 36, 8780–8785.
65 K. Horie, S. Yamada, S. Machida, S. Takahashi, Y. Isono and
H. Kawaguchi, Macromol. Chem. Phys., 2003, 204, 131–138.
66 R. Pelton, Adv. Colloid Interface Sci., 2000, 85, 1–33.
67 K. Iwai, Y. Matsumura, S. Uchiyama and A. P. de Silva, J. Mater.
Chem., 2005, 15, 2796–2800.
68 S. Uchiyama, N. Kawai, A. P. de Silva and K. Iwai, J. Am. Chem.
Soc., 2004, 126, 3032–3033.
48 B. Leng, L. Zou, J. B. Jiang and H. Tian, Sens. Actuators, B, 2009,
140, 162–169.
69 S. Uchiyama, Y. Matsumura, A. P. de Silva and K. Iwai, Anal. Chem.,
2003, 75, 5926–5935.
49 J. V. Ros-Lis, R. Martinez-Manez, K. Rurack, F. Sancenon, J. Soto
and M. Spieles, Inorg. Chem., 2004, 43, 5183–5185.
50 E. M. Nolan, M. E. Racine and S. J. Lippard, Inorg. Chem., 2006, 45,
2742–2749.
70 M. Q. Zhu, L. Y. Zhu, J. J. Han, W. W. Wu, J. K. Hurst and
A. D. Q. Li, J. Am. Chem. Soc., 2006, 128, 4303–4309.
71 Y. Shiraishi, R. Miyarnoto and T. Hirai, Langmuir, 2008, 24, 4273–
4279.
51 T. J. Dickerson, N. N. Reed, J. J. LaClair and K. D. Janda, J. Am.
Chem. Soc., 2004, 126, 16582–16586.
72 C. H. Li, Y. X. Zhang, J. M. Hu, J. J. Cheng and S. Y. Liu, Angew.
Chem., Int. Ed., 2010, 49, 5120–5124.
This journal is ª The Royal Society of Chemistry 2010
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