Fluorescent quenching of lanthanide-doped upconverting nanoparticles by photoresponsive polymer shells

Article abstract of DOI:10.1021/cm5021405

A photoresponsive amphiphilic polymer was synthesized and used to encapsulate upconverting lanthanide-doped nanoparticles to produce a novel water-dispersible nanoassembly with a high loading of emission quenchers. The nanoassembly exhibits fluorescent emission in the visible region upon irradiation with 980 nm light, which can be reversibly modulated by toggling the isomeric state of photoresponsive chromophores attached to the polymers backbone using UV and visible light. Photon counting experiments show that the quenching mechanism for this new nanoassembly is a combination of Foerster resonance energy transfer (FRET) and emission-reabsorption. Compared to the similar nanoassembly prepared from a reported plug-and-play method, this new nanoassembly has higher overall quenching efficiency due to the increased photoswitch loading (14 times compared to the existing nanoassembly).

Full text of DOI:10.1021/cm5021405

Article
pubs.acs.org/cm
Fluorescent Quenching of Lanthanide-Doped Upconverting
Nanoparticles by Photoresponsive Polymer Shells
Tuoqi Wu, Danielle Wilson, and Neil R. Branda*
4D LABS and Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A
1S6
S
* Supporting Information
ABSTRACT: A photoresponsive amphiphilic polymer was
synthesized and used to encapsulate upconverting lanthanide-
doped nanoparticles to produce a novel water-dispersible
nanoassembly with a high loading of emission quenchers. The
nanoassembly exhibits fluorescent emission in the visible region
upon irradiation with 980 nm light, which can be reversibly
modulated by toggling the isomeric state of photoresponsive
chromophores attached to the polymer’s backbone using UV
and visible light. Photon counting experiments show that the
quenching mechanism for this new nanoassembly is a
combination of Forster resonance energy transfer (FRET) and
̈
emission-reabsorption. Compared to the similar nanoassembly
prepared from a reported “plug-and-play” method, this new
nanoassembly has higher overall quenching efficiency due to the increased photoswitch loading (14 times compared to the
existing nanoassembly).
hile nanostructured fluorescent probes based on
ambient temperatures (both isomers are stable in the dark) and
can be subjected to a wide range of synthetic conditions to
tailor their optoelectronic properties, making them versatile
FRET acceptors.28−37
W
organic,1,2 silicon,3 carbon,4 and quantum dot5,6
materials offer many advantages in bioimaging applications,
their use would still benefit from improved image resolution.7
One approach to achieve this goal is to develop methods to
toggle a fluorescent probe between “light” emissive and “dark”
nonemissive states using an external stimulus.8−12 Light has
been used as the stimulus in imaging technologies such as
stimulated emission depletion (STED) microscopy,13 time-
gated STED microscopy,14 and stochastic optical reconstruc-
tion microscopy (STORM).15 Light is a particularly appealing
stimulus because it is noninvasive and can be easily tuned and
focused to provide a high degree of spatial and temporal
control. It has been successfully used to reversibly convert
photoresponsive chromophores between two isomeric forms,
The lanthanide-doped UCNPs38,39 have many advantages
over other emissive systems such as quantum dots. While the
use of quantum dots40−45 minimizes the photobleaching
observed for small molecule probes, it does not overcome the
problem of potential toxicity and “blinking” when used in
bioimaging applications. The former can be overcome by
modifying the surfaces of the quantum dots.46 But “blinking” is
still an unresolved issue.47,48 The lanthanide-doped UCNPs
absorb several photons of near-infrared (NIR) light and emit
them in the UV and visible region of the electromagnetic
spectrum.38,39 Because they absorb NIR light, which has deeper
tissue penetration and induces few organic photoreactions
(and, therefore, produces less damage to the surrounding
environment) than high energy UV or visible light, these
nanoparticles provide lucrative alternatives as emissive bio-
imaging probes.49 They also exhibit high photobleaching
resistance.50 These appealing features explain why we19−21
and others22,23,49 have developed several upconverting nano-
systems for potential use as fluorescent probes for in vitro and
in vivo imaging.
where only one of the isomers acts as an effective Forster
̈
resonance energy transfer (FRET) acceptor to quench the
fluorescence from nanoparticle donors.16−18
We19−21 and others22,23 have recently focused our attention
on developing variable-emission systems using dithienylethene
derivatives to decorate lanthanide-doped upconverting nano-
particles (UCNPs). These hybrid systems combine the
beneficial features of the photoresponsive chromophores and
the emissive nanoparticles. Dithienylethenes undergo reversible
cyclization reactions between colorless ring-open and colored
ring-closed isomers when exposed to UV and visible light
(Scheme 1)24−26 usually with a high degree of photofatigue
resistance during numerous ring-closing/ring-opening cycles.27
The members of this family of photoresponsive compounds
also tend to exhibit relatively good thermal bistability at
One of the challenges when developing systems that rely on
effective photochemical reactions is the fact that many of these
Received: June 12, 2014
Published: June 23, 2014
© 2014 American Chemical Society
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reactions operate poorly in aqueous conditions due to the poor
solubility or interaction between water and the chromo-
phores.51 This explains why we chose to carry out our initial
proof-of-concept studies in organic polymer films52 and organic
solvents.19,53 Our work in developing water-dispersible systems
for live organism imaging required considerable synthetic
efforts to modify the chromophores, attach them to the
nanoparticles (which had to be subjected to a ligand exchange
reaction prior to decorating them with the modified
chromophores), and retain the photoactivity of the chromo-
phore.20 These synthetic steps were avoided when we
demonstrated a “plug-and-play” method to prepare water-
dispersible nanosystems where both the UCNPs and the
photoresponsive diarylethenes were encapsulated by an
amphiphilic polymer.21 A simplified version of this system is
illustrated in Figure 1. The fact that both components reside
chromophores directly into the polymer used for wrapping
should increase the loading and emission quenching. This is the
focus of the studies reported in this paper.
EXPERIMENTAL SECTION
■
General Details. All solvents and reagents used for synthesis,
chromatography, UV−vis spectroscopy measurements, and photolysis
studies were purchased from Aldrich, Caledon, and Anachemia and
used as received, unless otherwise noted. Solvents for NMR analysis
were purchased from Cambridge Isotope Laboratories and used as
received. Column chromatography was performed using silica gel 60
(230−400 mesh) from Silicycle Inc. The NaYF4 nanoparticles
codoped with 2% Er3+ and 20% Yb3+ (NaYF4:ErYb) were synthesized
using a modification of a recently reported procedure.20 The
photochromic amine (1o) was synthesized as described in the
literature55 Poly(propylene glycol)bis(2-aminopropyl ether) (Jeff-
Amine 2000) was received as a gift from Huntsman Inc. Cumene
terminated poly(styrene-co-maleic anhydride) was purchased from
Aldrich. Amino functionalized PEG 2000 was purchased from Rapp
Polymere GmbH Inc. All volumes for absorption, photolysis, and
concentration measurements/studies were measured using an
autopipette.
Instrumentation. 1H NMR and 13C NMR characterizations of
synthetic precursors were performed on a Bruker BioSpin 400
spectrometer and were found to match the reported data. The
characterizations of the final compounds were performed on QNP 600
cryoprobe (proton sensitive, inverse probe) working at 600.33 MHz
for 1H and 150.97 MHz for 13C. The 1H NMR photolysis studies were
performed on CD3CN solutions using a Bruker BioSpin 500
spectrometer.
Optical Spectroscopy. UV−vis absorption spectroscopy was
performed using a Varian Cary 300 Bio spectrophotometer.
Fluorescence measurements were performed using a PTI Quanta-
master spectrofluorometer. A JDS Uniphase 980 nm laser diode
(device type L4-9897510-100M) coupled to a 105 μm (core) fiber was
employed as the excitation source. The output of the diode laser was
collimated and directed on the samples using a Newport F-91-C1-T
Multimode Fiber Coupler. The visible emissions were collected from
the samples at π/2 from the incident beam in the plane of the
spectrometer. All of the colloidal samples were held in a square quartz
cuvette with an interior width 4 mm and length 10 mm (Starna Cells,
Part # 9F/Q/10). All spectra were corrected for instrument sensitivity.
Transmission Electron Microscopy (TEM). TEM images were
obtained using a Hitachi 8100 scanning transmission electron
microscope operating at 200 keV. For the nanoparticles dispersed in
hexanes, a small amount of this dispersion was drop-cast on a carbon
Formvar-coated copper grid (400 mesh, Ted Pella, Part no. 01754-F)
and air-dried before imaging. The shape and size of the NaYF4:ErYb
nanoparticles were evaluated from the collected TEM images. The size
of the nanoparticles was calculated from over 50−70 particles located
at different areas of the TEM grid. For aqueous samples, dilute colloids
of the nanoparticles dispersed in water (5 μL) were placed on thin,
carbon Formvar-coated copper grids held by anticapillary tweezers
(Ted Pella, part no. 501-4). Water was then slowly removed under
reduced pressure in a vacuum desiccator.
Dynamic Light Scattering (DLS). DLS measurements were
carried out using a Malvern Zetasizer Nano-ZS. The colloidal samples
were held in a 10 mm path length plastic cuvette (BrandTech, Catalog
no. 759220). A nanoparticle concentration of ∼0.4 mg/mL was
employed for the measurements. All DLS measurements were
conducted at 25 °C.
Photoinduced Ring-Closing and Ring-Opening Reactions.
All photoreactions of the dithienylethene derivatives as small
molecules in solution (3o ⇄ 3c) or integrated in the nanoparticle
systems (2o-NP ⇄ 2c-NP and 3o(NP) ⇄ 3c(NP)) were monitored
using UV−vis absorption spectroscopy. All photoreactions were
carried out using the light source from a lamp used for visualizing
TLC plates at 365 nm (Spectroline E-series, 1.3 mW/cm2) to induce
the ring-closing reactions (3o → 3c, 2o-NP → 2c-NP, and 3o(NP) →
Figure 1. Simplified illustration of the nanosystem containing
upconverting nanoparticles wrapped in an amphiphilic polymer shell
containing trapped photoresponsive chromophores. Neither the
chromophores nor the nanoparticles have to be subjected to synthetic
modifications and ligand exchange using this approach.
within the hydrophobic “inner” shell created when the polymer
spontaneously wraps around the nanoparticles (1) eliminates
the need for ligand exchange of the oleate groups and
anchoring the photochromic molecules to the surface (although
we did demonstrate that attaching the photoresponsive
chromophores to the nanoparticles prior to wrapping them
with the polymer has the benefits of higher loading and more
effective UV photoswitching),54 (2) provides a method to
conveniently build a library of different systems by swapping
the organic chromophores, and (3) most importantly, creates a
local hydrophobic environment where photochemistry is not
diminished even in water.
While these systems represent effective examples to
demonstrate the “plug-and-play” concept and light-induced
reversible quenching of the emission from the nanoparticles,
their performance would have been substantially more
impactful if the loading of the chromophores was higher to
achieve a greater extent of fluorescence quenching. Our
assumption is based on the fact that, using the coencapsulation
method, only a small number of photochromic components
could be loaded into the hybrid system. Attaching the
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3c(NP)) and a tungsten filament light source with a greater than 490
nm cutoff filter (Elmo Omnigraphic 300AF, 107 mW/cm2) to induce
the ring-opening reactions (3c → 3o, 2c-NP → 2o-NP, and 3c(NP)
→ 3o(NP)). Monochromatic light was generated using a PTI
Quantamaster spectrofluorometer. The wavelength was adjusted to
be centered at 550 and 650 nm, and the slit width of the fluorometer
was adjusted to 1 nm in width.
for 25 min at 20600 RCF, and the supernatant was removed from the
pellets of nanoparticles using a pipet. The nanoparticles were
redispersed in deionized water (3 mL for each sample) with the
help of sonication, and the two samples were combined and passed
through a 0.2 μm filter (Acrodisc Syringe Filter) to obtain the final
stock solution of encapsulated nanoparticles for further use.
Synthesis of Diarylethene Amide 3o. A stirred solution of
amino-diarylethene 1o (57 mg, 0.1 mmol) in Et2O (5 mL) was treated
with acetic anhydride (20 μL, 22 mg, 0.22 mmol). The reaction
mixture was stirred at room temperature for 16 h, at which time it was
quenched by pouring it into 50 mL of saturated NaHCO3. The
mixture was extracted with Et2O (2 × 50 mL), and the organic layers
were combined, dried over MgSO4, filtered, and evaporated to dryness
using a rotary evaporator. Purification using column chromatography
(silica, hexanes:EtOAc 1:2, rf = 0.6) afforded 27 mg (47% yield) of
Fluorescence Lifetime Measurements. An aqueous solution of
2o-NP (1.17 × 10−4 M) or 3o(NP) (1.85 × 10−5 M) in a quartz
cuvette (Starna Cells, Part # 9F/Q/10) was excited with picosecond
laser pulses at 980 nm with an energy of 500 μJ/pulse. The two
concentrations reported here are total concentration of photoswitches
encapsulated in the nanoassemblies obtained using the concentration
of the ring-closed isomers of each photoswitch at its photostationary
state (calculated by UV−vis spectroscopy after baseline scattering
subtraction) divided by percentage of ring-closed isomers in this state
(measured by 1H NMR spectroscopy). The laser pulses were
generated by a picosecond optical parametric generator (OPG) from
EKSPLA, Model PG401. This OPG was pumped by an EKSPLA
PL2241 ps laser at 10 Hz repetition rate and 30 ps pulse duration. The
laser beam was focused to a 2 mm light column passing through the
sample cuvette along the 10 mm interior direction. The emission
spectral detection and lifetime measurements were performed by a
combination of spectrograph from Princeton Instruments, model
SP2300i, and streak camera system from Hamamatsu, model C7700.
The spectrograph used a 150 lines/mm grating providing 362 nm
spectral coverage centered at 532 nm, and the streak camera was
operated in photon counting mode with a temporal range of 500 μs.
The decay times were calculated from these curves by fitting them with
a single exponential model using Origin software (version 8.5) after
the in-growth portion of the curve.
Synthesis of Photoresponsive Polymer (2o). In a glass vial
equipped with a magnetic stir bar, a solution of diarylethene 1o (15
mg, 0.028 mmol) in CHCl3 (3 mL) was treated with vigorous stirring
with cumene terminated poly(styrene-co-maleic anhydride) (PSMA,
47.6 mg, 0.028 mmol, Mn = 1700). The vial was covered with
aluminum foil and the solution was stirred at room temperature for 20
h. The reaction mixture was cooled down to 0 °C and treated with
mPEG-NH2 (168 mg, 0.084 mmol, Mn = 2000), 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide (EDC, 16 mg, 0.084 mmol),
and triethylamine (17 mg, 0.168 mmol) all in one portion. The
reaction mixture was stirred at 0 °C for 1 h, allowed to warm to room
temperature, and stirred for an additional 20 h. The solvent was
removed using a rotary evaporator, and the oily residue was treated
with deionized water (3 mL). After sonication, the aqueous solution
containing the crude polymer product was dialyzed against deionized
water for 96 h in a dialysis bag with a molecular weight cutoff of 14
kDa (Spectrum Laboratories, Inc.). The purified polymer was isolated
by lyophilizing the resulting aqueous solution to dryness to afford 155
mg (34%) of polymer 2o as a green power. (The slight green color is
due to the small amount of ring-closing to generate some colored 2c.)
1H NMR (600 MHz, CD2Cl2) δ 7.17 (br, s, 65 H, phenyl protons of
PSMA), 3.63 (s, 701 H, OCH2CH2 of mPEG-NH2), 3.37 (s, 13H,
OCH3 of mPEG-NH2).
1
amide 3o as yellow crystals. Mp 208−210 °C. H NMR (600 MHz,
CDCl3) δ 7.55−7.52 (m, 4H), 7.49 (d, J = 8.4 Hz, 2H), 7.38 (t, J = 7.2
Hz, 2H), 7.30 (t, J = 7.2 Hz, 1H), 7.28 (s, 1H), 7.21 (s, 1H), 2.20 (s,
3H), 1.97 (s, 3H), 1.96 (s, 3H). 13C NMR (150 MHz, CDCl3) δ
168.4, 142.4, 141.9, 141.4, 141.1, 137.7, 136.2 (m, 2 carbons), 133.5,
129.6, 129.2, 128.1, 126.4, 126.0, 125.8, 122.5, 122.1, 120.3, 116.3 (tt, J
= 254, 24 Hz, 2 carbons), 111.0 (tq, J = 269, 24 Hz, 1 carbon), 24.8,
14.70, 14.6. (The splitting is due to coupling with the fluorine atoms
on the cyclopentene ring.) Anal. Calcd for C29H21F6NOS2: C, 60.30;
H, 3.66; N, 2.42. Found: C, 60.59; H, 3.48; N, 2.43. HRMS (M + H+)
expected: 578.1047; found: 578.1040.
Synthesis of Hybrid Nanosystem 3o(NP). In a typical synthesis,
a stirring solution of cumene terminated poly(styrene-co-maleic
anhydride) (PSMA, 25 mg, 14.6 μmol, Mn = 1700) in CHCl3 (3
mL) was treated with a solution of the oleate-coated UCNPs
(NaYF4:ErYb) (250 μL, 43 mg/mL) in CHCl3. After stirring at room
temperature for 2 h, the reaction was treated with a solution of
JeffAmine 2070 (150 mg, 80 μmol, Mn = 2070) in CHCl3 (1.0 mL)
and a stock solution (1.2 mM) of the photoresponsive compound 3o
(334 μL, 0.4 μmol) in CHCl3 using a pipet. The reaction was stirred at
room temperature for 16 h, at which time it was evaporated to dryness
using a rotary evaporator. The oily residue was treated with aqueous
NaOH (3 mL, 0.001 M, pH 11) and sonicated for 5 min, and any trace
amounts of CHCl3 were carefully removed using a rotary evaporator to
afford a clear aqueous solution. This solution was transferred using a
pipet into two 1.5 mL conical centrifugation tubes and centrifuged at
20600 RCF for 25 min. The supernatant was removed from the pellets
of nanoparticles using a pipet, and the pellets were redispersed in
deionized water (1.5 mL for each sample) with the help of sonication.
The tubes were centrifuged for 25 min at 20600 RCF, and the
supernatant was removed from the pellets of nanoparticles using a
pipet. The nanoparticles were redispersed in deionized water (1.5 mL
for each sample) with the help of sonication, and the two samples were
combined and passed through a 0.2 μm filter (Acrodisc Syringe Filter)
to obtain the final stock solution of encapsulated nanoparticles for
further use.
RESULTS AND DISCUSSION
■
In all reported examples, the amphiphilic polymer was prepared
by ring-opening the anhydride functional groups in copolymers
of maleic anhydride and hydrophobic alkenes with amines as
schematically shown in the top of Scheme 1. The previously
reported dithienylethene derivative bearing a single amino
functional group (1o)55 was chosen to prepare the polymers.
The dithienylethene amide 3o (Scheme 1) was prepared for
comparison by acetylating 1o.
Synthesis of Hybrid Nanosystem 2o-NP. In a typical synthesis,
a stirring solution of photoresponsive polymer 2o in CHCl3 (3 mL)
was treated with a solution of the oleate-coated UCNPs
(NaYF4:ErYb) in CHCl3 (375 μL, 43 mg/mL). This value is a
calculated value of the nanoparticle concentration based on the
volume and mass of air-dried aliquots sample of the original UCNPs in
CHCl3 solution. After stirring at room temperature for 20 h, the
reaction mixture was evaporated to dryness using a rotary evaporator.
The oily residue was treated with aqueous NaOH (3 mL, 0.001 M, pH
11), sonicated for 5 min, and any trace amounts of CHCl3 were
carefully removed using a rotary evaporator to afford a clear aqueous
solution. This solution was transferred using a pipet into two 1.5 mL
conical centrifugation tubes and centrifuged at 20600 RCF for 25 min.
The supernatant was removed from the pellets of nanoparticles using a
pipet, and the pellets were redispersed in deionized water (3 mL for
each sample) with the help of sonication. The tubes were centrifuged
The photoresponsive polymer 2o was prepared using a
modified procedure from our recent work54 and work
published by others.56−58 In a typical synthesis (Scheme 2),59
poly(styrene-co-maleic anhydride) (PSMA) was reacted with
1o in CHCl3. The resulting carboxylate was coupled to mPEG-
NH2 using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide
(EDC). The final polymer was isolated by removing the organic
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organic (3o) and inorganic components (NaYF4:ErYb) were
coencapsulated with a nonphotoactive amphiphilic polymer was
prepared using the original coencapsulation method21 as shown
in Scheme 3. This system was prepared to compare the loading
of the chromophores in the two systems as well as the effects of
loading on the fluorescence quenching.
Scheme 1. (top) Ring-Opening of Copolymers of Maleic
Anhydride with Amines to Produce Functionalized
Amphiphilic Polymers for Wrapping Upconverting
a
Nanoparticles and (bottom) Photoresponsive
Chromophores Used in These Studies Showing How They
Undergo Reversible Photochemical Reactions When
Exposed to UV and Visible Light
Dynamic light scattering (DLS) analyses of the nano-
assemblies show an average hydrodynamic size of 34.2 nm
for 2o-NP and 32.2 nm for 3o(NP).59 The similarity in the
diameters was supported by TEM imaging (Figure 2 and
Supporting Information Figure S1), which show average
diameter of 24 nm for 2o-NP and 23 nm for 3o(NP). The
smaller size of the nanoparticles measured using TEM
compared to DLS is expected as the former highlights the
inorganic core while the latter measures the hydrodynamic size
of the entire assembly including the polymer shell. What is also
revealed in the TEM images is a faint outline of the polymer
shell around the nanoparticles. The shell was estimated to be
approximately 3 nm thick, which correlates well with the
increased diameters of the nanoassemblies measured using DLS
techniques (Supporting Information Figure S2). An idealized
representation of the polymer shell with the mPEG chains fully
extended puts the maximum thickness of the coating at 10−11
nm, which is much greater than what was measured using either
TEM or DLS analysis. It is reasonable to assume the polymer
exists in a more compressed form, and it is unlikely that each
UCNP is encapsulated by more than one polymer layer.
The ring-closing and ring-opening reactions of all photo-
chromic systems (2o in 2o-NP, 3o in 3o(NP)) were monitored
using UV−vis absorption spectroscopy, where characteristic
bands in the visible region of the spectrum appear as is typical
for the ring-closing reactions of dithienylethene derivatives
when they are exposed to UV light (Figure 3, Supporting
Information Figure S3, and Table 1). In all cases, the high-
energy bands in the UV region (∼300 nm) decrease in intensity
as the colorless isomers (2o and 3o) are converted into their
colored ring-closed counterparts (2o and 3c) when solutions of
them (CH3CN for 3o and H2O for 2o-NP and 3o(NP)) are
exposed to 365 nm light. The bands are centered between 590
and 600, which explains why the solutions become blue.61 After
a
The black circle represents a hydrophobic group such as phenyl rings
or alkyl chains. The white circle is typically water dispersible chains
such as polyethylene glycols.
solvent, followed by dialysis against deionized water and
lyophilization. We estimate the ratio of the photochromic
component to the mPEG-2000 chain to be 1:4 according to the
1H NMR spectrum of 2o.60
Polymer 2o was used to encapsulate oleate-coated NaYF4
upconverting nanoparticles doped with 2% Er and 20% Yb
(NaYF4:ErYb) as shown in Scheme 2 by first mixing the two
components in CHCl3 followed by treatment with water to
induce encapsulation.59 The final assembly (2o-NP) was
isolated by centrifugation. Another nanosystem where both
Scheme 2. Synthesis of Photoresponsive Polymer and Encapsulation of Upconverting Nanoparticles
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Scheme 3. Synthesis of Nanosystems Containing Both Upconverting Nanoparticles and Dithienylethene 3o Simultaneously
Coencapsulated by an Amphiphilic, Non-Photoactive Polymer
Figure 2. Representative TEM images of the nanoassemblies used in
these studies. (a) Encapsulated system using the photoresponsive
polymer 2o-NP and (b) the system with coencapsulated components
(3o(NP)).
about 30−90 s of exposure to this light, the cyclization
reactions have reached their photostationary states (2o/c-NP,
3o/c, and 3o/c(NP)). In the case of the free chromophore
(3o/c), the amount of ring-closed isomer in the photosta-
tionary state was measured to be 75% by 1H NMR
spectroscopy. While the amounts of ring-closed isomers in
Figure 3. UV−vis absorption spectra of H2O solutions of (a) 2o-NP
1
the nanoassemblies are impossible to measure directly by H
NMR spectroscopy, we estimated them to be similar to 3o/c
(1.17 × 10−4 M) and (b) 3o(NP) (1.85 × 10−5 M),63 before (solid)
and after (broken line) irradiation with 365 nm light. The green and
based on the fact that there are almost no observed differences
between the all absorbance spectra for systems when they are
normalized at the λmax (300 nm) for the ring-open isomers
(Supporting Information Figure S3d). Based on this
assumption, we calculated the average loading of the
chromophores per nanoparticle in the assemblies to be 1212
for 2c-NP and 87 for 3c(NP).62 As anticipated, the number of
photoresponsive components significantly increased when they
were directly conjugated into the polymer used to wrap the
nanoparticles, and this will have a consequence on the ability of
the systems to quench the emission from the UCNPs.
red shaded areas in each absorption spectrum correspond to the region
where the UCNPs emit when irradiated with 980 nm light to show the
spectral overlap. (c) Normalized emission spectra (λex = 980 nm) of
2o-NP before irradiation with 365 nm light (white shaded) and at
photostationary state (dark green and red shaded) and 3o(NP) before
irradiation with 365 nm light (white shaded) and at photostationary
state (light green and red shaded). (d) Relative emission of 2o-NP
before irradiation with 365 nm light (white shaded) and at
photostationary state (dark green and red shaded) and 3o(NP)
before irradiation with 365 nm light (white shaded) and at
photostationary state (light green and red shaded).
When any of the colored solutions containing the photosta-
tionary states are exposed to visible light of wavelengths greater
than 490 nm, they all revert to their colorless states as the ring-
open isomers are regenerated. The original spectra are also
reproduced. These ring-closing/ring-opening cycles can be
repeated numerous times with minimal observable changes in
the spectra.
Figure 2 also illustrates how the chromophores in the
nanoassemblies can reversibly modulate the emission from the
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Table 1. Absorption Characteristics, Photochemistry, and Loading of the Chromophores in Nanoassemblies 2o-NP and 3o(NP)
optical properties
ring-open isomer
λ (nm)
ε (cm−1 M−1
ring-closed isomer
c
photoresponsive system
)
λ (nm)
ε (cm−1 M−1
)
photostationary state (%)
number of chromophores per nanoparticle
a
3o/c
300
304
303
61882
−
−
591
595
600
26175
−
−
75
−
1212
b
d
2o/c-NP
−
b
d
3o/c(NP)
−
87
a
b
c
d
In CH3CN. In H2O, after correcting for baseline scattering. As measured using 1H NMR spectroscopy. These values are assumed to be the same
as for the free chromophore (3o/c) in solution.
Table 2. Emission Characteristics and Fluorescence Lifetimes for 2o-NP and 3o(NP)
a
b
relative emission
fluorescent lifetime (μs)
FRET quenching (%)
per chromophore
system
505−570 nm
625−690 nm
ring-open isomer
ring-closed isomer
total
26
3.9
2o/c-NP
0.35 0.02
0.85 0.03
0.15 0.01
0.76 0.02
50.4 0.7
51.9 0.5
37.1 0.9
49.9 0.5
1
1
0.021 0.001
0.045 0.01
3o/c(NP)
a
Ratio of the area under the curve in the emission spectrum of the nanosystems after irradiation with 365 nm light until the photostationary states
were reached to the area under the curve in the emission spectrum of the nanosystems prior to irradiation (2o-NP and 3o(NP)). The mean values
b
and standard deviations are based on six ring-closing/ring-opening cycles. Obtained by using an exponential fit of the photon-counting data. The
errors are from the fitting uncertainty. The data were collected only from 505 to 570 nm due to the weak signal between 625 and 690 nm under
optimized conditions.
upconverting nanoparticles. When 980 nm light is used as the
excitation source, both nanosystems (2o-NP and 3o(NP))
show similar emission characteristics. This is expected
considering there is minimal spectral overlap between the
emission bands of the nanoparticles and the absorption bands
of the ring-open isomers (2o and 3o) so there should be
minimal effect from either chromophore no matter what the
level of loading. As is typical for NaYF4:ErYb nanoparticles,39
two sets of relatively sharp bands are observed in the region
between 500 and 700 nm. The higher energy “green” emissions
chromophore in 3o(NP)). The “red” quenching shows similar
results (0.07% and 0.28%, respectively). These differences are
somewhat unexpected given their similar absorption spectra
and can be attributed to the chromophores in 3o(NP) residing
closer to the nanoparticles. This conclusion is supported by
lifetime measurements as discussed in the next section of this
report.
The two major mechanisms responsible for the quenching of
emission from the nanoparticles are Forster resonance energy
̈
transfer (FRET) and “emission-reabsorption”.65 The former
involves discrete energy donors and acceptors that transfer the
excited state energy through collisions.66 The latter is often
ignored. In this case, quenching is a result of the light emitted
from the nanoparticle being absorbed by chromophores in
close proximity but not necessarily anchored to the nano-
particle that is emitting the light. Although the net observable
effect is the same, the FRET mechanism is important to
enhance when quenching is required in many environments of
use. One good example is bioimaging applications, where the
concentration of the emissive probe at any one time and at any
one place is too low to rely on the contribution of “emission-
reabsorption”. Because FRET is independent of concentration
in a hybrid system containing both “donor” and “acceptor”
intimately linked it will still operate. In our previous examples
of photoresponsive upconverting nanoparticles we provided
evidence that the major contribution to the quenching was
“emission-reabsorption”, and FRET was only responsible for
7−26% of the quenching.21 This was the incentive to prepare
and study the photoresponsive polymer systems reported in
this paper (2o-NP).
4
4
(504−568 nm) correspond to the [4H11/2, S3/2] → I15/2
transitions, while the lower energy “red” emissions (627−684
4
nm) are a result of the F9/2
→
4I15/2 transitions. Both
transitions overlap with the absorption band of the ring-closed
isomer in the photostationary states (2c-NP and 3c(NP)),
which is shown by the reduced amount of emission from either
nanosystem when it is irradiated with 365 nm light (Figure 3c).
Because the ring-closed isomers integrated in both nano-
assemblies have similar absorption profiles, both “green” and
“red” nanoparticle emissions are reduced, albeit to different
extents (Figure 3c,d and Table 2).64 As anticipated, visible light
(>490 nm) restores the original fluorescence intensity since it
triggers the ring-opening reactions and regenerates the isomers
that do not effectively quench the emission for the nano-
particles. As in the case of the absorption changes, the emission
quenching can be subjected to several ring-closing/ring-
opening cycles without observable degradation.
The nanoassembly that has the photochromic component
directly conjugated into the wrapping polymer (2o-NP) is over
four times more effective at quenching the “green” emission as
the assembly that has the photochromic component
coencapsulated (3o(NP)). It is three times as effective for
the “red” emission. Both observations can be attributed to the
increased loading of the fluorescence quencher in the
nanoassembly (2o-NP has almost 14 times the number of
photochromic components as 3o(NP) as shown in Table 1).
However, if the relative “green” quenching is considered per
chromophore in the two nanoassemblies, it is clear that the
chromophores in 3o(NP) are more effective quenchers (0.05%
quenching per chromophore in 2o-NP vs 0.17% quenching per
Fluorescence lifetime measurements for both nanoassemblies
show a more significant increase in the “green” emission decay
kinetics67 for 2o-NP → 2o/c-NP (from 50 to 37 μs) when
compared to 3o(NP) → 3o/c(NP) (from 52 to 50 μs). These
data are listed in Table 2. These values correspond to FRET
efficiencies of 26% for 2o/c-NP and 4% for 3o/c(NP). Again,
the argument for a greater extent of FRET quenching due to
higher chromophore loading in 2o/c-NP can be used.
However, if the relative amounts of FRET quenching per
chromophore are compared (0.021% quenching per chromo-
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Chemistry of Materials
Article
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phore in 2o-NP vs 0.045% quenching per chromophore in
3o(NP)), it is once again clear that the chromophores in the
latter system are more effective quenchers. Because the FRET
mechanism is highly dependent on the relative orientation and
proximity of the acceptor to the donor, the freedom the
chromophores exhibit in 3o(NP) results in their finding more
suitable positions. In the case of 2o-NP the chromophores are
anchored to the polymer backbone, which limits their mobility.
The focus of this paper is the enhanced (but still reversible)
fluorescence quenching of nanoassemblies that have the
photoresponsive chromophore directly integrated into the
polymer used to wrap upconverting nanoparticles. While
attaching the chromophores as pendant groups onto the
polymer backbone somewhat reduces the modular “plug-and-
play” approach to make libraries of these systems, the increase
in loading makes them attractive. We believe these systems will
help in the design of the next generation of organic−inorganic
hybrid nanoassemblies for use in bioimaging applications. The
fluorescent probe we developed has the potential to enhance
the spectral resolution of fluorescent microscopy. Future
research will focus on increasing loading while allowing
mobility of the chromophores to enhance FRET quenching
in UCNP−diarylethene hybrid system.
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ASSOCIATED CONTENT
* Supporting Information
Details on the materials for synthesis, instrumentation for
analysis, and microscopy and spectral characterizations. This
material is available free of charge via the Internet at http://
pubs.acs.org.
■
(17) Klajn, R.; Stoddart, J. F.; Grzybowski, B. A. Chem. Soc. Rev.
2010, 39, 2203.
S
(18) The focus of this report is on the use of photoresponsive
systems to modulate the emissive properties of nanoparticles. The
numerous examples in which photochromic compounds and light have
been used to control the fluorescence and phosphorescence of small
molecules have not been included in this paper. We encourage the
reader to refer to several illustrative reviews on this subject. (a) Cusido,
J.; Deniz, E.; Raymo, F. M. Eur. J. Org. Chem. 2009, 2031. (b) Raymo,
F. M.; Tomasulo, M. Chem. Soc. Rev. 2005, 34, 327.
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2012, 10, 6159.
AUTHOR INFORMATION
Corresponding Author
*E-mail: nbranda@sfu.ca (N.R.B).
Author Contributions
■
(20) Boyer, J. C.; Carling, C. J.; Chua, S. Y.; Wilson, D.; Johnsen, B.;
Baillie, D.; Branda, N. R. Chem.Eur. J. 2012, 18, 3122.
(21) Wu, T.; Boyer, J.-C.; Barker, M.; Wilson, D.; Branda, N. R.
Chem. Mater. 2013, 25, 2495.
(22) Zhou, Z.; Hu, H.; Yang, H.; Yi, T.; Huang, K.; Yu, M.; Li, F.;
Huang, C. Chem. Commun. 2008, 4786.
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
(23) Zhang, C.; Zhou, H.; Liao, L.; Feng, W.; Sun, W.; Li, Z.; Xu, C.;
Fang, C.; Sun, L.; Zhang, Y.; Yan, C. Adv. Mater. 2010, 22, 633.
(24) Feringa, B. L. Molecular Switches; Wiley-VCH: Weinheim, 2010.
(25) Tian, H.; Yang, S. Chem. Soc. Rev. 2004, 33, 85.
(26) Ubaghs, L.; Sud, D.; Branda, N. R. Handbook in Thiophene-Based
Materials: Applications in Organic Electronics and Photonics; Perepichka,
I. D., Perepichka, D., Eds.; John Wiley & Sons: Chichester, 2009; Vol.
2.
(27) Other classes of photochromic compounds tend to show less
stability than dithienylethenes when integrated into emissive nano-
particle systems. Yildiz, I.; Impellizzeri, S.; Deniz, E.; McCaughan, B.;
Callan, J. F.; Raymo, F. M. J. Am. Chem. Soc. 2011, 133, 871. Although
even some dithienylethenes undergo degradation. Erno, Z.; Yildiz, I.;
Gorodetsky, B.; Raymo, F. M.; Branda, N. R. Photochem. Photobiol. Sci.
2010, 9, 249.
ACKNOWLEDGMENTS
■
This research was supported by the Natural Sciences and
Engineering Research Council (NSERC) of Canada, the
Canada Research Chairs Program, and Simon Fraser
University. This work made use of 4D LABORATORIES
shared facilities supported by the Canada Foundation for
Innovation (CFI), British Columbia Knowledge Development
Fund (BCKDF), and Simon Fraser University. We thank Dr.
Saeid Kamal for performing photon counting experiments and
providing useful suggestions.
ABBREVIATIONS
NP, nanoparticle; Mp, melting point; HRMS, high resolution
mass spectroscopy
■
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(29) Giordano, L.; Jovin, T. M.; Irie, M.; Jares-Erijman, E. A. J. Am.
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1
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