Lanthanide-based luminescent probes for selective time-gated detection of hydrogen peroxide in water and in living cells

Article abstract of DOI:10.1039/c0cc01560a

Lanthanide-based luminescent probes TPR1 and TPR2 were developed for the detection of hydrogen peroxide (H₂O₂) in living systems. The chemoselective reaction of these boronate-protected probes with H ₂O₂ resulte

Full text of DOI:10.1039/c0cc01560a

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Lanthanide-based luminescent probes for selective time-gated detection of
hydrogen peroxide in water and in living cellsw
Alexander R. Lippert, Tina Gschneidtner and Christopher J. Chang*
Received 25th May 2010, Accepted 7th August 2010
DOI: 10.1039/c0cc01560a
Lanthanide-based luminescent probes TPR1 and TPR2 were
developed for the detection of hydrogen peroxide (H2O2) in
living systems. The chemoselective reaction of these boronate-
protected probes with H2O2 resulted in an enhanced lanthanide
sensitization and a 6-fold increase in luminescent intensity.
TPR2 was utilized to measure the endogenous production of
H2O2 in RAW 264.7 macrophages using time-gated luminescent
spectroscopy.
excited states. Elegant examples of lanthanide–antenna
complexes have been reported for luminescent detection of
pH,15 metal ions,16 reactive oxygen species,17 bone fissures,18
and assorted metabolites,19 where the majority of these probes
use binding events to physically alter the coordination
environment of the lanthanide center for sensing purposes.
We sought to pursue an alternative approach where electronic
modulation of lanthanide–antenna interactions could be used
as a sensing strategy and now present the synthesis and
characterization of a new class of lanthanide-based luminescent
probes for H2O2 that operate through a reaction-based
sensitizer mechanism. Terbium Peroxy Reporters 1 and 2
(TPR1 and TPR2) feature high selectivity for detecting H2O2
over an array of biologically relevant ROS in water and in
living cells in a time-gated mode by long-lived lanthanide
emission.
Reactive oxygen species (ROS) comprise a family of small
molecules that play a variety of important roles in human
health, aging, and disease.1–10 In particular, H2O2 maintains a
privileged position amongst these oxygen-derived metabolites
owing to its chemical versatility as well as relative stability
in living systems.2 Controlled H2O2 bursts in response to
physiological stimuli can regulate beneficial cellular processes,
including growth and proliferation,3 differentiation,4 migration,5
and phagocytosis,6 whereas misregulation of H2O2 chemistry
in the cell can lead to pathological states that contribute to
aging7 and diseases ranging from cancer8 to diabetes9 to
neurodegenerative disorders.10 The signaling/stress dichotomy
of H2O2, along with its small size and transient nature in
complex biological environments, provides significant challenges
for disentangling its physiological and/or pathological
contributions to healthy and disease states.
Our general strategy for time-gated luminescence detection
of H2O2 relies on exploiting the chemoselective H2O2-mediated
oxidation of a boronate protecting group to uncage a pendant
aniline or phenol antenna group for sensitizing lanthanide
emission (Scheme 1). In the absence of H2O2, the boronate-
capped aromatic is electron-withdrawing and a poor lanthanide
sensitizer; reaction with H2O2 generates an electron-rich
phenol or aniline antenna group capable of transferring energy
to a complexed lanthanide ion and give a turn-on luminescent
response (Scheme 1). This overall design is inspired by
previous reports that tyrosine can sensitize Tb3+ and Eu3+
luminescence20 and that an amide/aniline switch can be used
for time-resolved detection of protease activity.21 In particular,
we targeted the DO3A-based platforms incorporating a boronate
cage linked directly to the masked sensitizer or through a self-
immolative carbamate. Full synthetic routes to TPR1 and
We have initiated a program aimed at studying the
chemistry and biology of H2O2 by molecular imaging and
have reported fluorescent indicators for chemoselective
11
detection of H2O2 and their use in understanding H2O2
fluxes generated in response to growth factor,11e ion channel,12
and immune response activation.11f,j With the goal of expanding
these efforts to other imaging approaches, we were attracted to
the advantageous properties of luminescent lanthanide
complexes, particularly in terms of minimizing background
interference from biological specimens.13 The long emission
lifetimes of lanthanide ions allow the use of time-gated
techniques, which can eliminate background from short-lived
autofluorescence of native cellular species,14 while large
apparent Stokes shifts and narrow emission bands for lanthanide
complexes can reduce self-quenching and other complications
from scattered light.14 Because lanthanides exhibit low molar
extinction coefficients, they require sensitization with organic
ligand antennas capable of energy transfer to lanthanide
Department of Chemistry and the Howard Hughes Medical Institute,
University of California, Berkeley, CA 94720, USA.
E-mail: chrischang@berkeley.edu; Fax: +1 510 642 7301;
Tel: +1 510 642 4704
w Electronic supplementary information (ESI) available: Synthetic,
spectroscopic, and bioassay details. See DOI: 10.1039/c0cc01560a
Scheme 1 Terbium Peroxy Reporters 1 and 2 (TPR1 and TPR2).
c
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TPR2 are provided in the ESI.w The phenol (TbÁ3) and aniline
(TbÁ4) products resulting from H2O2 oxidation were also
independently synthesized and characterized.
We evaluated the spectroscopic properties of TPR1 and
TPR2 and their responses to H2O2 in aqueous solution
buffered to physiological pH (20 mM HEPES, pH 7.4). Spectra
were acquired after a 100 ms delay, effectively suppressing back-
ground fluorescence from the organic sensitizer. TPR1 exhibits
a maximum absorbance centered at 226 nm (e = 6700 MÀ1 cmÀ1
)
for the boronate antenna with weak lanthanide emission
(F = 0.029, t = 1.26 ms). TPR2 has a more intense absorption
for the boronate-capped aryl carbamate sensitizer (240 nm,
e = 15 000 MÀ1 cmÀ1) but is even more dim in terms of
lanthanide luminescence (F = 0.006, t = 1.36 ms). Although,
the absolute absorbance maxima for these complexes occur at
240 nm and 226 nm, the largest differences in the excitation
spectra between the probes and their oxidative products occur
near 280 nm (Fig. S5, ESIw), due to the unmasking of a phenol
(TPR1) or aniline (TPR2) which have absorbance maxima
near this wavelength. Treatment of both probes with H2O2
triggers a patent increase in long-lived luminescence intensity
with concomitant formation of phenol (TbÁ3) and aniline
(TbÁ4) products (Fig. 1). The newly-synthesized phenol product
Fig. 2 Time-gated, luminescent turn-on responses of TPR1 and
TPR2 to biologically relevant ROS. All ROS were added at a
concentration of 200 mM except for O2À, which was enzymatically
generated at 6 mM minÀ1 to give a final concentration of either 360 or
720 mM. (a) Bars represent relative emission intensities at lem = 545 nm
at 0 (white), 30 (light grey), 60 (grey), 90 (dark grey), and 120 (red) min
after ROS addition to 10 mM TPR1. (b) Bars represent relative
emission intensities at lem = 545 nm at 0 (white), 15 (light grey), 30
(grey), 45 (dark grey), and 60 (red) min after ROS addition to 10 mM
TPR2. Data were acquired at 25 1C in 20 mM HEPES, pH = 7.4, with
excitation at lex = 280 nm and dÀelay and gate times of 100 ms and
10 ms, respectively, except for O2 assays, which were measured in
50 mM phosphate buffer at pH 7.8 for enzyme compatibility. TBHP =
tert-butyl hydroperoxide.
immune response. The cells were then washed and endogenous
H2O2 production was measured by time-gated luminescence
spectroscopy (Fig. 3). Whereas the steady-state emission
spectrum is dominated by autofluorescence and scattered
light, completely swamping any signal from the lanthanide
luminescence (Fig. 3a, dotted black trace), the time-gated
spectrum displays distinct bands characteristic of long-lived
emissive terbium complexes (Fig. 3a, solid red trace). We
observe a ca. 10% increase in time-gated TPR2 emission in
PMA-stimulated macrophages compared to control cells
(Fig. 3b), establishing that TPR2 is capable of detecting
changes in H2O2 levels in living cells by sensitized lanthanide
luminescence.
TbÁ3 possesses an absorption at 226 nm (e = 10 200 MÀ1 cmÀ1
)
with long-lived lanthanide emission (F = 0.054, t = 1.23 ms);
these values are comparable to the known aniline TbÁ4
complex (F = 0.051, t = 1.47 ms).21 We observe a faster
comparative H2O2 response for TPR2 over TPR1, as the
former attains the same 6-fold turn-on enhancement in half
the time. We then tested the selectivity of TPR1 and TPR2 by
screening their reactivities against a panel of physiologically
relevant ROS (Fig. 2). Both reporters show highly specific
turn-on luminescent responses for H2O2 over these reactive
oxygen and/or nitrogen metabolites.22
The foregoing results establishing that TPR1 and TPR2 can
selectively respond to H2O2 in water by a lanthanide-sensitized
luminescence increase led us to apply these tools for detecting
H2O2 produced in living cells by time-gated emission spectro-
scopy. We utilized the TPR2 probe for these studies owing to
its faster relative turn-on response. Live RAW 264.7 macro-
phages were loaded with 10 mM TPR2 and activated with
1 mg mLÀ1 phorbol myristate acetate (PMA) for 6 hours at
37 1C to stimulate H2O2 generation through a phagocytic
In conclusion, we have developed a pair of turn-on
luminescent lanthanide probes for H2O2 in water and in living
cells. TPR1 and TPR2 utilize a reaction-based antenna switch
to trigger a selective emission increase from a pendant long-lived
Fig. 3 Time-gated luminescence detection of H2O2 produced in living
cells using TPR2. (a) Emission spectra of TPR2 in live RAW 264.7
macrophages. The dotted black trace displays the steady-state
emission spectrum with lex = 280 nm. The solid red trace shows the
time-gated emission spectra with lex = 280 nm, using a delay time =
100 ms and a gate time = 10 ms. (b) Graph showing relative time-gated
luminescence intensities of RAW 264.7 macrophages incubated for
6 hours at 37 1C with 10 mM TPR2 (control) or 10 mM TPR2 and
1 mg mLÀ1 PMA (PMA). Data were acquired with lex = 280 nm, and
delay and gate times of 100 ms and 10 ms, respectively.
Fig. 1 Time-gated, luminescent turn-on responses of 10 mM TPR1
and TPR2 to 200 mM hydrogen peroxide. (a) Responses of TPR1 at 0,
30, 60, 90, and 120 min after H2O2 was added. (b) Responses of TPR2
at 0, 15, 30, 45, and 60 min after H2O2 was added. Data were acquired
at 25 1C in 20 mM HEPES, pH = 7.4, with excitation at lex = 280 nm
and delay and gate times of 100 ms and 10 ms, respectively.
c
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lanthanide center. TPR2 is capable of detecting elevations in
H2O2 production in live macrophages during oxidative
immune response using time-gated luminescence spectroscopy,
showing that this approach can deliver optical probes that
minimize background fluorescence from interfering organic
species in biological samples. Whereas time-gated imaging is
possible using the UV excitation of these first-generation
probes, visible excitation is preferred to minimize cellular
damage. Additionally, quantitative H2O2 analysis would
require precise kinetic information of the boronate oxidation
reaction as well as measurement of the local probe concentration.
We are currently pursuing next-generation ratiometric
lanthanide complexes with improved sensitizer switches and
longer wavelength visible excitations to optimize turn-on
responses for time-gated imaging of H2O2, as well as expanding
the concept of electronic modulation of antennae for developing
new long-lived lanthanide-based optical and MRI probes.
This work was supported by the National Institutes of
Health (GM 79465), Amgen, Astra Zeneca, Novartis, and
the Packard and Sloan Foundations. C.J.C. is an Investigator
with the Howard Hughes Medical Institute.
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