Multiplex detection of enzymatic activity with responsive lanthanide-based luminescent probes

Article abstract of DOI:10.1002/anie.201408560

Multiplex analyte detection in complex dynamic systems is desirable for the investigation of cellular communication networks as well as in medical diagnostics. A family of lanthanide-based responsive luminescent probes for multiplex detection is reported. The high modularity of the probe design enabled the rapid assembly of both green and red emitters for a large variety of analytes by the simple exchange of the lanthanide or an analyte-cleavable caging group, respectively. The real-time three-color detection of up to three analytes was demonstrated, thus setting the stage for the non-invasive investigation of interconnected biological processes. © 2015 Wiley-VCH Verlag GmbH & Co. KGaA , Weinheim.

Full text of DOI:10.1002/anie.201408560

Angewandte
Chemie
DOI: 10.1002/anie.201408560
Fluorescent Probes
Multiplex Detection of Enzymatic Activity with Responsive
Lanthanide-Based Luminescent Probes**
Elias Pershagen and K. Eszter Borbas*
Abstract: Multiplex analyte detection in complex dynamic
systems is desirable for the investigation of cellular commu-
nication networks as well as in medical diagnostics. A family of
lanthanide-based responsive luminescent probes for multiplex
detection is reported. The high modularity of the probe design
enabled the rapid assembly of both green and red emitters for
a large variety of analytes by the simple exchange of the
lanthanide or an analyte-cleavable caging group, respectively.
The real-time three-color detection of up to three analytes was
demonstrated, thus setting the stage for the non-invasive
investigation of interconnected biological processes.
T
he reliable determination of enzymatic activity is crucial for
fundamental cell biology and biochemistry, drug develop-
ment, diagnosis, and therapeutic follow-up.[1] As enzymatic
activity is dependent on the proteinꢀs expression level,
localization, microenvironment, and substrate as well as
cofactor availability, it is most accurately measured directly
in situ.[2] The ability to monitor the activities of multiple
enzymes in real-time, potentially in conjunction with the
concentrations of regulators, would be immensely valuable.
Information from such experiments would reduce the risk of
false-positive or -negative diagnoses, facilitate the profiling of
both healthy and diseased cells, and could establish connected
nodes in cellular communication networks. We present
a palette of lanthanide (Ln) based turn-on luminescent
probes that enable the simultaneous, two-color detection of
an enzyme and either a small molecule or a second enzyme. In
combination with an organic-based fluorophore, the three-
color detection of two enzymes and a regulatory small
molecule was achieved. The well-separated Ln emissions
make data interpretation and real-time analyte detection
straightforward (Figure 1a).
Figure 1. a) Comparision of the absorption and emission spectra of
Ln- and organic-based emitters. b) Design of the turn-on probe.
be detected simply by altering the chemoselectively cleavable
cage. Probes for b-glucosidase (b-Glu), b-galactosidase (b-
Gal), a-mannosidase (a-Man), and phosphatase were built
(Scheme 1). Analytes were chosen due to their importance as
reporter enzymes,[4] in disease diagnosis,[5] in drug develop-
ment,[5,6] and in biochemical assays.[7] A boronate-caged
probe for reactive oxygen and nitrogen species (RONS;
[8]
H2O2 and ONOOÀ [9]) was also prepared. RONS are
essential for cellular communication.[10] Protein tyrosine
phosphatases (PTPs) can be regulated by RONS upon
oxidation of their catalytically active Cys residue.[11] The co-
detection of phosphatase activity and [RONS] has potential
ramifications for drug discovery.[12] While Ln-based enzyme
probes are known, these are either not able to detect
enzymatic activity in real time,[13] require potentially damag-
ing high-energy excitation,[14] or have low brightness.[3]
Crucially, methods (luminescent or other) for the real-time
measurement of enzymes and their regulators (e.g. PTPs and
RONS[15]) are currently lacking.
Ln emission in these probes is turned-on by the formation
of a light-harvesting antenna from a caged precursor (Fig-
ure 1b).[3] The design is highly modular, as a new analyte can
[*] E. Pershagen, Dr. K. E. Borbas
Department of Chemistry—BMC, Uppsala University
Box 576, Uppsala, 75123 (Sweden)
E-mail: eszter.borbas@kemi.uu.se
[**] This work was supported by the Swedish Research Council (project
grant 2013-4655) and the Department of Chemistry—BMC. We
thank Prof. Gunnar von Heijne (Department of Biochemistry and
Biophysics, Stockholm University) for access to a plate reader, Dr.
Julien Andres for help with phosphorescence and quantum yield
measurements as well as critical reading of the manuscript, and Dr.
Sashi Vithanarachchi and Prof. Mikael Widersten for critical reading
of the manuscript.
The responsive probe LnCou (“on”) and model com-
pound LnBn (“off”) were prepared in short, high-yielding
syntheses (see the Supporting Information). Photophysical
characterization was carried out on the model compounds
(Table 1, see Figures S2–S5 in the Supporting Information).
LnBn and LnCou had absorption maxima at 335 and 350 nm,
respectively. The Ln excitation spectra were similar to their
absorption spectra, thereby confirming Ln sensitization by
the antennae. Excitation was possible with near-UV light,
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201408560.
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Scheme 1. Probes used in this work.
Table 1: Photophysical characterization.
Figure 2. a) Time-resolved emission spectra of EuBn and EuCou in
H2O. b) Turn-on response of Eu probes recorded on a plate reader.
The emission is normalized to those of the caged complexes.
[Complex]=50 mm; [enzyme]=5 U b-Glu, 1 U a-Man, 0.25 U b-Gal,
0.1 U phosphatase; 258C; lex =356 nm; 250 ms delay; 1050 ms sample
window; 100 mm HEPES, pH 7.0 or pH 8.0 (EuPhos). HEPES=2-[4-(2-
hydroxyethyl)-1-piperazinyl]ethanesulfonic acid.
Entry[a]
lmax
[nm]
e[a]
[mÀ1 cmÀ1
FLn
FL [%]
tH O
tD O
q[d]
[a]
[a,b]
2
2
]
[ms][a]
[ms][c]
EuCou
TbCou
GdCou
EuBn
TbBn
GdBn
350
350
350
335
335
335
13240
13016
15280
7580
4540
4920
1.08 (37)
1.63 (32)
– (33)
0.12 (0.9)
0.41 (0.7)
– (0.6)
1.233
0.724
–
1.165
0.971
–
3.500
0.774
–
3.375
0.689
–
0.33
0.15
–
0.37
À2.41
–
ure 2b, see also Figures S6–S12 in the Supporting Informa-
tion). These responses were 81- and 90-fold higher for EuCou/
EuBn and TbCou/TbBn, respectively, which are among the
highest observed for Ln-based turn-on probes.[21] An over 50-
fold increase in the Ln emission was recorded for the b-Gal
probe EuGal, and a 20–36-fold increase for EuPhos, EuMan,
and EuGlu with alkaline phosphatase, a-Man, and b-Glu,
respectively. Progress curves of different enzyme concentra-
tions plotted versus time ꢁ [enzyme] were non-superimpos-
able,[22] thus suggesting eventual deactivation of the enzyme,
presumably caused by the quinone methide by-product.[23]
The EuBpin response was linear for 0–100 mm [H2O2] (see
Figure S11 in the Supporting Information).
One of the most attractive features of Ln-based probes is
the interchangeability of metals, which results in probes with
different emission colors: yellow (Dy), green (Tb), orange
(Sm), red (Eu), and near-infrared (Yb, Nd). This streamlines
the development of probes for multicolor and ratiometric
detection.[24] The latter is advantageous due to its independ-
ence from probe concentration and bleaching. Ln-complex
mixtures can afford a ratiometric readout if the ratio of the
antennaꢀs sensitization ability for the Ln changes during the
experiment.[25] The addition of analyte to Eu- and Tb-probe
mixtures resulted in an increase in the intensity ratio of the
618 nm/545 nm emission (see Figures S13 and S14 in the
Supporting Information). The rate of this increase provides
a measure of the in vitro enzyme activity. This method is
[a] In H2O. [b] With coumarin 2[18] or Cs3[Ln(dpa)3][19] as reference.
dpa=dipicolinic acid. [c] In D2O. [d] Calculated as described in Ref. [20].
with a cut-off at approximately 380 nm. Such low-energy
excitation is desirable for biological applications. The LnBn
complexes showed weak residual fluorescence of the antenna
(lem = 409 nm), which was intensified and red-shifted (lem
=
457 nm) in LnCou. The lowest-energy triplet of GdBn was at
23500 cmÀ1, with a lifetime at 77 K of (194 Æ 8) ms, while that
of GdCou was found at 21000 cmÀ1, and had a long lifetime at
77 K of (1028 Æ 74) ms. The low-temperature lifetime of the
triplet state shortened in EuCou (299 ms) and TbCou
(616 ms). These preliminary data do not enable the assign-
ment of either a triplet[16] or a singlet-mediated[17] Ln-
sensitization pathway. The TbCou emission was slightly
sensitive to oxygen, probably because of energy back transfer
from the Tb excited state to the coumarin triplet. An increase
in the Tb lifetime to 2.62 ms at 77 K, and a 1.8-fold increase in
the Tb emission upon deoxygenation support this proposal.
However, the presence of oxygen did not interfere appreci-
ably with analyte detection. The hydration numbers of q < 1
are typical of octadentate complexes. The negative q value of
TbBn could be explained by multiple deactivation pathways.
To evaluate the turn-on response afforded by the probes,
the integrated emission increases were determined (Fig-
2
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complementary to other Ln-based ratiometric detection
strategies.[26]
antenna. Three non-interacting analytes (b-Gal, alkaline
phosphatase, H2O2) could be monitored independently of
each other at 450, 545, and 655 nm, respectively (Figure 3a).
Two interacting analytes (PTP1B, H2O2) were then studied
together with a non-interacting analyte (b-Gal). The EuGal
emission depended only on b-Gal. When H2O2 was present,
the intensity of the TbPhos signal diminished compared to
that in the absence of H2O2, thus reflecting the inactivation of
PTP1B by H2O2. The CouBpin emission is essentially
unaffected by PTP1B, which enables the accurate determi-
nation of [H2O2] (Figure 3b). Several chromophores are
competent antennae for 5[28]–7[29] different lanthanides, which
suggests that this three-color detection could be further
extended with alternative antennae.
In conclusion, a suite of responsive luminescent Ln
complexes was prepared by exploiting analyte-triggered
formation of an antenna. The probes have high stabilities in
the absence of their analytes and excellent turn-on character-
istics. By virtue of their modularity and ease of synthesis, they
can be readily adapted to the detection of various analytes.
The simultaneous detection of two analytes was straightfor-
ward without spectral cross-talk between the emitters.
Furthermore, the Ln-based probes could be combined with
an organic-based fluorophore with absorption and emission
wavelengths outside of the probesꢀ range to achieve three-
color analyte detection. It is worth noting that all the
experiments could be performed on a bench-top fluorimeter
or a plate reader, and data analysis was simple due to the well-
separated signals. The methods reported herein represent
a significant step forward in multiplex detection in complex
biological systems. The expansion of the probe palette and the
use of these complexes for the investigation of cellular
communication networks are ongoing.
Nonresponsive Ln complexes are well established for
multicolor labeling.[27] However, multicolor analyte detection
by the use of responsive Ln emitters has only recently become
possible.[3] Tools for the real-time, simultaneous monitoring of
enzymes with their small-molecule or enzyme regulators
could greatly facilitate the mapping of cellular information
flow. Our probes were created with such applications in mind,
and in the following experiments their utility was explored.
First, we established that we could detect two enzymes
simultaneously by using probes with different emission wave-
lengths (see Figures S15 and S16 in the Supporting Informa-
tion). It was also possible to co-detect a regulatory small
molecule/enzyme pair,[11,15] specifically the RONS-dependent
deactivation of human PTP1B, while measuring [H2O2] (see
Figure S17 in the Supporting Information). Finally, the
possibility of three-color detection was explored. Two Ln
emitters were used in combination with an organic fluoro-
phore,
a
RONS-responsive 7-pinacolborane coumarin
(CouBpin, Scheme 1). H2O2-mediated oxidation of the bor-
onate to afford the phenol yields blue fluorescence, which
does not overlap with the Ln emissions. CouBpin is excitable
at 410 nm, where the Ln complexes do not absorb appreci-
ably, thus eliminating interfering fluorescence from the
Received: August 26, 2014
Revised: October 21, 2014
Published online: && &&, &&&&
Keywords: enzymes · fluorescent probes · lanthanides ·
.
luminescence · reactive oxygen species
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Fluorescent Probes
Getting real: The simultaneous, real-time
detection of two enzymes or an enzyme
and a small molecule by time-resolved
luminescence spectroscopy has been
demonstrated using lanthanide-based
responsive probes. The use of two differ-
ent lanthanide emitters results in well-
separated output signals (see picture),
while three-color analyte detection with-
out spectral overlap can be achieved by
combination with an organic fluorophore-
based probe.
E. Pershagen,
K. E. Borbas*
&&&& — &&&&
Multiplex Detection of Enzymatic Activity
with Responsive Lanthanide-Based
Luminescent Probes
Angew. Chem. Int. Ed. 2014, 53, 1 – 5
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!