Caged quantum dots

Article abstract of DOI:10.1021/ja804948s

Photoactivatable organic fluorophores and fluorescent proteins have been widely adopted for cellular imaging and have been critical for increasing temporal and spatial resolution, as well as for the development of superresolution microscopy techniques. At

Full text of DOI:10.1021/ja804948s

Published on Web 11/05/2008
Caged Quantum Dots
Gang Han,†,‡ Taleb Mokari,‡ Caroline Ajo-Franklin,† and Bruce E. Cohen*,†
Biological Nanostructures and Inorganic Nanostructures Facilities, The Molecular Foundry, Lawrence Berkeley
National Laboratory, Berkeley, California 94720
Received June 27, 2008; E-mail: becohen@lbl.gov
Photoactivatable organic fluorophores and fluorescent proteins
(FPs) have been widely adopted for cellular imaging and have been
critical for increasing temporal and spatial resolution,1,2 as well as
for the development of superresolution microscopy techniques.3,4
Semiconducting nanocrystal quantum dots (QDs) possess superior
brightness and photostability compared to either organic fluoro-
phores or FPs but have not yet been engineered for microscopic
photoactivation. As part of our efforts to develop nanoparticles with
novel optical properties, we have synthesized photocaged quantum
dots, which are nonluminescent under typical microscopic illumina-
tion but can be activated with stronger pulses of UV light.
ortho-Nitrobenzyl (ONB) groups have classically been used to
cage biomolecules, rendering them inactive until pulsed with light.5,6
UV irradiation induces a photolytic reaction, freeing the parent
molecule along with a nitrosocarbonyl byproduct. With QDs, certain
Figure 1. Quantum dot uncaging with o-nitrobenzyl ligands.
aromaticgroupshavepreviouslybeenfoundtoquenchluminescence,7,8
and we sought to determine whether ONB groups could do so until
released photochemically (as shown in Figure 1).
Scheme 1
CdTe/CdS core/shell QDs were grown under aqueous conditions
in the presence of mercaptopropionic acid (MPA),9,10 and lipoic
acid-derived dithiolate ligands11 were synthesized to contain an
ONB-phosphoryl group. The phosphoryl group has proved to be
an excellent substrate for caging groups6 and possesses good
aqueous solubility. The 4,5-dimethoxy-1-(2-nitrophenyl)ethyl (DM-
NPE) caging group12 absorbs at the longer UV wavelengths (ca.
355-413 nm) commonly used to photoswitch fluorescent proteins
and produces a nitrosoketone byproduct (Figure 1) less toxic than
those of other caging groups.
Mixtures of compound 3 (synthesized as shown in Scheme 1)
and its noncaged analogue 4 were added to the nanoparticles and
allowed to fully displace the monothiol MPA. Exchange occurred
very rapidly, judging from an obvious loss of luminescence within
the first few seconds upon mixing (not shown). This method of
MPA displacement on water-grown QDs permits facile placement
of varying percentages of caging groups onto the QD surface, as
confirmed by their absorbance spectra13 (Figure 2a). MPA displace-
ment also permits the addition of other ligands for bioconjugation,
cell compatibility, and targeting.15
ONB byproduct from the QD surface (Figure 2b-d). Noncaged
QDs also typically displayed an increase in PL quantum yield upon
UV exposure, though much smaller (ca. 1.3-fold) than that for the
caged QDs, consistent with previous reports of photobrightening
effects caused by the annealing of surface traps.9,17 Longer
illumination times led to full restoration of 3-caged QD lumines-
cence (Figures 2d and S2a), and at the longest times we consistently
and unexpectedly observed caged CdTe QDs become brighter than
their noncaged counterparts exposed to the same conditions.
Photolysis also caused a small decrease in the size of these QDs in
solution, from ca. 7 to 6 nm, as measured by dynamic light
scattering (Figure S1).
Green CdTe/CdS QDs (λmax ) 520 nm) coated with 25% caged
compound 3 showed a ca. 400-fold reduction in PL quantum yield
compared to identical QDs coated with noncaged 4 (Figures 2b
and S2a). By comparison, the difference between dark and bright
states of PA-GFP is ca. 100-fold,2 similar to the “fully quenched”
state of dopamine-coated CdSe/ZnS QDs.7 This contrast ratio is
critical to imaging applications that require significant differences
between dark and bright states.4,16
Exposure of caged QDs to 2 mW/mm2 365-nm light produced
an increase in PL, as would accompany photolytic release of the
We synthesized a second lipoic acid derivative 5 (Chart S1),
with the ONB held fewer atoms from the QD surface than in
† Biological Nanostructures Facility.
‡ Inorganic Nanostructures Facility.
9
10.1021/ja804948s CCC: $40.75
2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 15811–15813 15811

C O M M U N I C A T I O N S
Figure 2. Optical characterization of QDs with varying percentages of surface ligand 3. For all QDs, the remaining ligand is compound 4. (a) Absorption
spectra of green CdTe/CdS QDs and surface ligand alone. (b) PL spectra of CdTe/CdS QDs with ONB ligand 3, excited at 405 nm. QD spectra are normalized
to the absorbance at the first exciton. (c) PL spectra of QDs in Figure 2b following 2 min of 365-nm irradiation. (d) PL spectra of QDs in Figure 2b following
10 min of irradiation.
Figure 3. Transient PL emission detected with TCSPC, following 440-nm excitation of (a) green CdTe/CdS QDs (λmax ) 525 nm) and (b) red CdTe/CdS
QDs (λmax ) 625 nm).
compound 3. QDs coated with 5 showed consistently lower PL
yields than QDs coated with similar fractions of 3 (Figure S2a),
suggesting that the ONB-QD distance affects quenching efficiency.
These QDs also underwent PL increases upon illumination, but less
efficiently than QDs coated with 3, possibly owing to the relatively
poor uncaging ability of thiols compared to the phosphates.
We also sought to understand how ONB interacts with QDs of
different compositions and emissions. Green InP/ZnS QDs (ref 14,
λmax ) 524 nm) were quenched 30-fold by a 25% 3 surface coating
(Figure S2b), about an order of magnitude less than comparable
green CdTe/CdS QDs, possibly due to differences in shell thickness.
Red CdTe/CdS QDs (λmax ) 625 nm) also displayed quenching by
surface-bound ONB, though to a significantly lesser degree than
the green QDs (Table S1). For near-infrared (nIR) CdHgTe/ZnS
QDs (ref 17, λmax ) 760 nm), quenching was lesser still, about
25-fold, even with a 100% surface coating (Table S1). For all QDs,
quenching increased with increasing fractions of surface ONB,
although this effect saturated at a certain fraction. Importantly, we
observe that ONB cages QDs over a wide spectral window, from
green into the nIR.
photons before they can reach the nanocrystal. We examined red
CdTe/CdS ONB-coated QDs excited at the first exciton (Figure
S2c), where ONB has no measurable absorbance (Figure 2a). These
QDs still showed a decrease in PL quantum yield, but this decrease
is less than that found with 405-nm excitation (Figure S2d). This
reveals some inner filter effect, but the observed quenching with
605-nm excitation suggests the primary effect arises from an
opticoelectronic coupling of the ONBs with the nanocrystal.
We therefore examined these red and green QDs with time-
correlated single-photon counting spectroscopy (TCSPC) to deter-
mine if ONB affects the exciton lifetime (Figure 3). PL lifetimes
of caged QDs were shorter than noncaged QDs, and the mean PL
decay time decreased with increasing fraction of surface ONB,
suggesting that ONB acts as a dynamic quencher of the exciton
state. As with steady-state PL, this effect was more pronounced
for QDs of shorter wavelength emissions. These shortened lifetimes
are indicative of an electron or hole transfer from QD to ONB,7,18
particularly since there is no overlap between QD emission and
ONB absorption (Figure 2), excluding the possibility of exciton
energy transfer.
One possible mechanism of ONB quenching would involve an
inner filter effect, in which surface-bound ONB groups absorb
To determine the cellular compatibility of caged QDs and their
photolysis, we incubated green CdTe/CdS QDs coated with 10%
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15812 J. AM. CHEM. SOC. VOL. 130, NO. 47, 2008

C O M M U N I C A T I O N S
of Basic Energy Sciences, Division of Materials Sciences and
Engineering, of the U.S. Department of Energy under Contract No.
DE-AC02-05CH11231.
Supporting Information Available: Experimental procedures and
figures, a chart, and a table. This material is available free of charge
via the Internet at http://pubs.acs.org.
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Figure 4. Live-cell imaging of QD uncaging. (a) Brightfield image of NIH
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3. Luminescent images of cells (b) before and (c) after photolysis with 387-
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identically with (a)-(c). Scale bar is 10 µm
ligand 3 with murine fibroblasts (Figure 4), which have previously
been shown to endocytose QDs.19 The remaining surface ligand
was a mixture of positive and negative charges (compounds 6 (Chart
S1) and 4, respectively), which has been shown to minimize
nonspecific QD adhesion to cell surfaces.15 Before UV irradiation,
little luminescence above cellular autofluorescence is visible
(compare Figures 4b and 4e). Photolysis through the objective using
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docytosed caged QDs (Figure 4c), but not control cells (Figure 4f).
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In summary, we have endowed quantum dots with one of the
more useful properties of bioimaging probes: the ability to be
switched on with light. The ONB caging group efficiently quenches
QD luminescence and can be released from the nanoparticle surface
with UV light. Caging is dependent on the emission of the QD but
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large array of new colors for photoactivatable probes. Like
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nm2 surface area. For 80 dithiolates, each will have a footprint of 0.15
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Acknowledgment. We thank Tracy Mattox for technical support
and Carolyn Bertozzi, Brett Helms, Jim Schuck, and Ron Zuck-
ermann for comments on the manuscript. Work at the Molecular
Foundry was supported by the Director, Office of Science, Office
JA804948S
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