Reversible three-state switching of multicolor fluorescence emission by multiple stimuli modulated FRET processes within thermoresponsive polymeric micelles

Article abstract of DOI:10.1002/anie.201002203

Micellar traffic lights: The title FRET system (FRET=fluorescence resonance energy transfer) consists of one type of donor dye and two types of acceptor dyes. On/off fluorescence switching of the latter two dyes can be controlled by pH changes and light, respectively. This novel type of multicolor luminescent polymeric assembly can act as a ratiometric probe for pH and temperature with tunable sensitivity. (Figure Presented).

Full text of DOI:10.1002/anie.201002203

Communications
DOI: 10.1002/anie.201002203
Luminescence Probes
Reversible Three-State Switching of Multicolor Fluorescence Emission
by Multiple Stimuli Modulated FRET Processes within
Thermoresponsive Polymeric Micelles**
Changhua Li, Yanxi Zhang, Jinming Hu, Jianjun Cheng,* and Shiyong Liu*
Energy transfer between light-absorbing donors and energy-
receiving acceptors occurs on the nanometer distance scale.[1]
One of the key issues in designing effective multichromo-
phore luminescent systems is the precise spatial arrangement
of the fluorophores. By learning from elegant systems existing
in nature, researchers have exploited a variety of artificial
nanostructures such as dendrimers,[2] nanoparticles,[3] (multi-
layered) thin films,[4] and supramolecular assemblies[5] to
achieve nanoscale control and accurate location of chromo-
phores, leading to the modulation of luminescence efficiency
through the enhancement or restriction of fluorescence
resonance energy transfer (FRET) processes. In view of the
microenvironment complexity in certain bio-applications
such as imaging, biosensing, and clinical diagnosis, it is
highly desirable to combine the concept of external stimuli-
triggered activation/deactivation of specific emitting fluoro-
phores to achieve higher temporal and spatial detection
resolution. Although there are a few examples of luminescent
polymeric assemblies and nanoparticles exhibiting two-state
switching of luminescence,[6] systems exhibiting both rever-
sible three-state on/off switching of the fluorescence emission
and stimuli-responsive tuning of the spatial distributions of
FRET donors and acceptors (FRET efficiency) has, to our
knowledge, not been accomplished.
cesses (Scheme 1). The FRET system consists of one type of
donor dye and two types of acceptor dyes, and fluorescence
emission of the latter two can be switched on and off by
changes in pH and light irradiation (UV/Vis), respectively.
Such multicolor luminescent polymeric assemblies can act as
sensitive ratiometric probes for pH and temperature. Most
importantly, the detection sensitivity can be further improved
at elevated temperatures because of the closer proximity
between FRET donors and acceptors resulting from the
thermoresponsive collapse of micelle coronas.
Three polymerizable fluorescent dyes, NBDAE, pH-
switchable rhodamine B based monomer (RhBAM; synthe-
sis: Scheme S1, NMR data: Figure S1 in the Supporting
Information), and photoswitchable SPMA, were synthe-
sized.[7] Amphiphilic diblock copolymer, P(St-co-NBDAE-
co-SPMA)20-b-P(NIPAM-co-RhBAM)60, bearing NBDAE
and SPMA moieties in the hydrophobic polystyrene (PS)
block and RhBAM moieties in the thermoresponsive poly(N-
isopropylacrylamide) (PNIPAM) block was synthesized by
sequential reversible addition–fragmentation transfer
(RAFT) polymerization (Scheme S2, Figures S2 and S3 in
the Supporting Information).[8] For comparison, a series of
PS-b-PNIPAM diblock copolymers with varying combina-
tions of NBDAE, SPMA, and RhBAM residues were also
synthesized. The molecular parameters of all diblock copoly-
mers used in this work are summarized in Table S1 in the
Supporting Information.
Herein we report the fabrication of amphiphilic and
thermoresponsive diblock-copolymer-based luminescent
micelles exhibiting three-state switchable multicolor fluores-
cence emission by external stimuli-modulated FRET pro-
In aqueous solution, P(St-co-NBDAE-co-SPMA)20-b-
P(NIPAM-co-RhBAM)60 self-assembles into spherical
micelles, as evidenced from AFM results (Figure S4 in the
Supporting Information), which consist of PS cores embedded
with NBDAE and SPMA dyes and thermoresponsive
PNIPAM coronas embedded with RhBAM dyes.[9] Surface
tensiometry measurements at 258C revealed a critical micelle
concentration (CMC) of 2.3 ꢀ 10ꢀ3 gLꢀ1 (Figure S5 in the
Supporting Information). The micellar solution at a concen-
tration of 1.0 gLꢀ1 exhibits thermo-induced aggregation
above 288C owing to the well-known lower critical solution
temperature (LCST) phase-transition behavior of PNIPAM
coronas (Figure S6 in the Supporting Information).[10]
Dynamic laser light scattering (LLS) analysis further revealed
intensity-average hydrodynamic diameters hDhi of 50 nm and
36 nm for the micellar solution at 258C and 358C, respectively
(Figure S7 in the Supporting Information). A comparison of
AFM and dynamic LLS analysis results indicated the
shrinkage of the thickness of the micellar coronas from
approximately 18 to 11 nm upon heating above the LCST of
PNIPAM coronas.
[*] C. Li, Y. Zhang, J. Hu, Prof. Dr. S. Liu
CAS Key Laboratory of Soft Matter Chemistry
Hefei National Laboratory for Physical Sciences at
Microscale Department of Polymer Science and Engineering
University of Science and Technology of China
Hefei, Anhui 230026 (China)
Fax: (+86)551-360-7348
E-mail: sliu@ustc.edu.cn
Prof. Dr. J. Cheng
Department of Materials and Engineering
University of Illinois at Urbana-Champaign
1304 West Green Street, Urbana, IL 61801 (USA)
Fax: (+1)217-333-2736
E-mail: jianjunc@illinois.edu
[**] The financial support of the National Natural Scientific Foundation
of China (NNSFC) Project (20874092) and the Specialized Research
Fund for the Doctoral Program of Higher Education (SRFDP) is
gratefully acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201002203.
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tion). The photoswitchable fluorescence emission behavior of
SPMA has been well-documented.[6a–c,12] SPMA exists in the
colorless spiropyran form under visible light; upon UV
irradiation, it transforms from the nonfluorescent form into
the merocyanine form (MCMA), which has fluorescence
emission at 645 nm (Scheme 1). Considering that the fluores-
cence emission of both NBDAE and MCMA can be enhanced
when located in hydrophobic microenvironment,[7a,12] we
choose to copolymerize NBDAE and SPMA moieties into
the hydrophobic PS block and pH-sensitive RhBAM dyes
into the thermoresponsive PNIPAM block.
Interestingly, the NBDAE fluorescence emission band
overlaps well with the absorbance bands of ring-opened
RhBAM and MCMA moieties (Figures S8 and S10 in the
Supporting Information). Thus, they can be integrated into
the diblock copolymer to construct efficient luminescent
systems with FRET features.[6b,c,13] P(St-co-NBDAE-co-
SPMA)20-b-P(NIPAM-co-RhBAM)60 possesses one type of
donor dye (NBDAE) and two types of potential FRET
acceptor dyes, RhBAM and SPMA. The fluorescence emis-
sion of the latter two can be switched on and off by pH and
light irradiation (UV/Vis), respectively. This novel type of dye
combination might lead to the construction of three-state
switchable multicolor luminescent systems [NBDAE emis-
sion at pH 7 under visible light, NBDAE/RhBAM(open
form) FRET system below pH 6 under visible light, and
NBDAE/MCMA FRET system at pH 7 upon UV irradia-
tion]. Moreover, the efficiency of the FRET process between
NBDAE and ring-opened RhBAM below pH 6 can be facilely
tuned by temperature variations by exploiting the thermo-
induced collapse of PNIPAM coronas, which results in the
closer proximity between FRET donors and acceptors
(Scheme 1).
We then explored the pH-modulated luminescence
behavior of P(St-co-NBDAE-co-SPMA)20-b-P(NIPAM-co-
RhBAM)60 micellar solution; under the initial conditions,
SPMA moieties in the PS block are colorless and non-
fluorescent. Above pH 6, only the green fluorescence emis-
sion of NBDAE dyes (lem = 518 nm, Figure 1A) is observed,
as RhBAM residues exhibit no emission, that is, a FRET
process between NBDAE and RhBAM or SPMA does not
occur. Upon adjusting to pH < 6, RhBAM moieties convert
into the fluorescent ring-opened form, as evidenced from the
appearance of a new fluorescence emission band at around
580 nm (Figure 1). Figure 1C clearly shows that the pH
decrease in the range pH 3–6 results in an increase of
emission intensity (580 nm) of ring-opened RhBAM, accom-
panied by a considerable decrease of NBDAE emission
intensity at 518 nm. The pH-induced fluorescence emission
changes can also be visualized by the naked eye, as evidenced
from the transition from green emission at pH 7 to yellow
emission at pH 3 (Figure 1B). The above results indicated the
occurrence of a FRET process between NBDAE within
micellar cores and ring-opened RhBAM moieties within
micellar coronas below pH 6, as the emission of NBDAE
residues in diblock copolymer micelles is essentially inde-
pendent of solution pH (Figure S8 in the Supporting Infor-
mation). When the solution pH was cycled between pH 7 and
3, we observed repeated changes of emission intensity at
Scheme 1. Construction of a polymeric-micelle-based reversible three-
state switchable multicolor luminescent system from amphiphilic and
thermoresponsive diblock copolymer P(St-co-NBDAE-co-SPMA)-b-
P(NIPAM-co-RhBAM). See text for details.
Among the three copolymerized dyes, NBDAE exhibits
strong green fluorescence emission at 520 nm, the intensity of
which is almost irrespective of solution pH in the range pH 3–
9 (Figure S8 in the Supporting Information).[7a] The pH-
sensitive RhBAM moiety exists in the spirolactam form above
pH 6 and exhibits no fluorescence emission, whereas below
pH 6 it transforms into the “open” form and emits strong
fluorescence at 580 nm (Scheme 1).[11] Thus, the fluorescence
emission of RhBAM residues can be facilely switched on and
off by pH variations (Figure S9 in the Supporting Informa-
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obtained for dual dye-labeled diblock copolymers, P(St-co-
NBDAE)20-b-P(NIPAM-co-RhBAM)60 and P(St-co-
NBDAE-co-SPMA)20-b-PNIPAM60, as control samples (Fig-
ures S12–S14 in the Supporting Information). The above
results indicated that the micellar solution of P(St-co-
NBDAE-co-SPMA)20-b-P(NIPAM-co-RhBAM)60 can act as
sensitive ratiometric pH probes in the range pH 3–6 (Fig-
ure 1C and Figure S15 in the Supporting Information). We
further employed the thermo-induced collapse of PNIPAM
coronas to adjust spatial proximity between NBDAE and
ring-opened RhBAM moieties located within micellar cores
and coronas, respectively (Scheme 1). As shown in Fig-
ure 1A,B, heating the micellar solution from 258C to 358C
leads to a yellow-to-orange transition of the fluorescence
emission. We can clearly observe the considerable increase of
RhBAM emission and the dramatic decrease of NBDAE
emission at pH 3 and 358C, relative to that at pH 3 and 258C.
This result indicated the occurrence of a more-efficient FRET
process for diblock copolymer micelles with collapsed
PNIPAM coronas. A more-detailed investigation revealed
that emission intensity ratios I580/I518 exhibit around a 3.5
times increase in the narrow temperature range 27–328C
(Figure 2a and Figure S16 in the Supporting Information).
Thus, the diblock copolymer micelles can also act as
ratiometric fluorescent thermometers. From Figure S15 and
Figure 1. A) Fluorescence emission spectra (lex =470 nm, slit widths
for excitation and emission: 5 nm) and B) photograph recorded
immediately (unless otherwise stated) under irradiation at 365 nm by
a UV lamp for micellar solutions of P(St-co-NBDAE-co-SPMA)20-b-
P(NIPAM-co-RhBAM)60 under various conditions: a) pH 7 and 258C;
b) pH 3 and 258C; c) pH 3 and 358C; and d) pH 7 and 258C upon UV
irradiation for 2 min. C) Fluorescence emission spectra under the
same conditions in the pH range 3–9.
580 nm, indicating that pH-induced emission switching is fully
reversible (Figure S11 in the Supporting Information).
Under visible-light conditions, the presence of SPMA
moieties in the diblock copolymer does not exhibit any
appreciable effects on the pH-switchable luminescence
behavior, as evidenced from spectrofluorimetric results
Figure 2. A) Fluorescence intensity ratio changes (I580/I518; conditions
as before) as a function of temperature recorded for the micellar
solution (pH 3) of P(St-co-NBDAE-co-SPMA)20-b-P(NIPAM-co-
RhBAM)60. B) Time evolution of fluorescence spectra (conditions as
before) recorded for the micellar solution (258C and pH 7) upon UV
(365 nm) irradiation.
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water was replaced approximately every 6 h. The micellar solution
Figure 2a it follows that the diblock copolymer micelles can
act as fluorescent ratiometric dual probes for pH and
temperature. Moreover, the detection sensitivity of pH can
be considerably enhanced at 358C, relative to that at 258C
(Figure S15).
exhibited no macroscopic phase separation upon standing at room
temperature for more than three months, suggesting the formation of
stable micelles. All pH adjustments were made by adding aqueous
HCl or NaOH.
The presence of SPMA moieties in P(St-co-NBDAE-co-
SPMA)20-b-P(NIPAM-co-RhBAM)60 diblock copolymer can
add extra features to the modulation of multicolor emission.
We further investigated the photoswitchable emission behav-
ior of the diblock copolymer micelles at pH 7 and 258C, under
which conditions RhBAM moieties exhibit no fluorescence
emission; thus, only the green emission from NBDAE
moieties can be observed under visible light (Figure 1B).
We observed the appearance of a new emission band at
around 645 nm upon UV irradiation, the intensity of which
gradually increased with irradiation time and stabilized out
after 1 min (Figure 2b and Figure S17 in the Supporting
Information). Concomitantly, the NBDAE emission intensity
at 518 nm exhibits a dramatic decrease. This change indicates
the occurrence of a FRET process between NBDAE and the
merocyanine form MCMA. Visual inspection of the micellar
solution under a UV lamp (365 nm) revealed an abrupt green-
to-red transition in fluorescence emission (Figure 1B). It is
notable that under UV irradiation at pH 7 and 258C, RhBAM
residues are stable and exist in the closed form, as evidenced
from the control sample only containing NBDAE and
RhBAM dyes (Figure S18 in the Supporting Information),
which might be ascribed to the chemical structure and the fact
that RhBAM moieties are located in a hydrophilic micro-
environment.[14] Upon five cycles of alternate UVand visible-
light irradiation, the emission intensity at 645 nm can
essentially recover to its original value (Figure S17 in the
Supporting Information).
In conclusion, we demonstrated the first example of an
amphiphilic responsive block copolymer micelle based multi-
chromophore luminescent system exhibiting reversible three-
state switching of fluorescence emission (green, yellow,
orange, and red) by modulating two independent FRET
processes by external stimuli (e.g., pH, temperature, and light
irradiation). The reported diblock copolymer micelles can
serve as sensitive ratiometric fluorescent dual probes to pH
and temperature; moreover, the detection sensitivities can be
facilely adjusted through thermo-induced collapse of respon-
sive micellar coronas owing to the closer proximity between
the FRET donors and acceptors. This novel type of multicolor
luminescent polymeric assemblies augurs well for practical
applications in cell imaging, biosensing, and clinical diagnosis.
Received: April 14, 2010
Published online: June 22, 2010
Keywords: block copolymers · fluorescence · fluorescent probes ·
.
FRET · micelles
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Experimental Section
Experimental details, including the synthesis procedures, character-
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in the Supporting Information.
Preparation of micellar solutions: P(St-co-NBDAE-co-SPMA)20-
b-P(NIPAM-co-RhBAM)60 (10 mg) was dissolved in N,N-dimethyl-
formamide (DMF; 1 mL). Under vigorous stirring, deionized water
(9 mL) was added through a syringe pump at a flow rate of
0.2 mLminꢀ1. After the addition was completed, the dispersion was
left to stir for another 5 h. DMF was then removed by dialysis (MW
cutoff, 14000 Da) against deionized water for 24 h. Fresh deionized
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