Signal ratio amplification via modulation of resonance energy transfer: Proof of principle in an emission ratiometric Hg(II) sensor

Article abstract of DOI:10.1021/ja066144g

Boradiazaindacene dyads designed as energy transfer casettes can be modified to signal cation concentrations ratiometrically. If the energy transfer efficiency is increased via changing spectral overlap on cation binding, an enhancement of emission signal

Full text of DOI:10.1021/ja066144g

Published on Web 10/21/2006
Signal Ratio Amplification via Modulation of Resonance Energy Transfer:
Proof of Principle in an Emission Ratiometric Hg(II) Sensor
Ali Coskun and Engin U. Akkaya*
Department of Chemistry, Middle East Technical UniVersity, Ankara, TR-06531, Turkey
Received August 30, 2006; E-mail: akkayaeu@metu.edu.tr
Fluorescent chemosensor design is an active field of supramo-
lecular chemistry, not only because of potential practical benefits
in cell physiology, analytical, and environmental chemistry,1 but
also as a proving ground for manipulation and/or engineering of
various photophysical processes toward an ultimate goal of selective
and sensitive signaling of targeted molecular or ionic species.2
Recently, boradiazaindacenes have become the fluorophore of
choice in many chemosensor designs,3 not only because of their
exceptional properties as fluorophores, but also as a result of their
remarkably rich chemistry.4 Previously, we reported3d a dimeric
boradiazaindacene, which can be converted into an energy transfer
cassette and furthermore into a ratiometric ICT (internal charge
transfer) based cation sensor, selective for silver ions, all through
simple structural modifications. In that design the two fluorophores
were kept very close to each other, so that the through-space EET
was nearly 100% efficient, thus creating a large pseudo-Stokes’
shift chemosensor. ICT based chemosensors typically have an
advantage of two distinct emissive states (analyte-free and analyte-
bound), which makes these chemosensors potentially wavelength-
ratiometric, that is, internal referencing of the signal is possible,
eliminating potential artifacts.
Figure 1. Structure of the boradiazaindacene dyads and the reference
compound 1.
In designing ratiometric chemosensors, one of most important
parameters is the magnitude of the range of signal ratios of emission
intensities at two different wavelengths for the analyte-free and
analyte-bound chemosensor, preferably excited at the isosbestic
point. This parameter determines the dynamic range and the
sensitivity of the chemosensor to the analyte concentration.5 A large
range of ratios could be obtained, only when the emission peaks
for the bound and free chemosensor are well-resolved and if both
forms have reasonable emission intensities. However, this ratio is
an inherent property of the particular chemosensor-analyte interac-
tions, and, until now, a systematic methodology for improving the
signal ratio was unavailable. We now introduce a new strategy in
ratiometric chemosensor design which would make this possible:
the range of ratios can be significantly improved, if the chemosensor
is designed as an energy transfer dyad, and once the interchro-
mophoric distance is carefully adjusted, binding of the analyte
increases the spectral overlap between the donor emission and the
acceptor absorption peaks. Thus, more efficient Fo¨rster type energy
transfer in the bound state results in higher emission intensity for
the analyte-bound chemosensor, effectively increasing the signal
ratio for the two states.
To demonstrate the feasibility of this approach, we set out to
synthesize a series of boradiazaindacene dyads, with increasing
interchromophoric distance. In chemosensors in which the metal
receptor is an electron donor and also part of the π-system of the
chromophore, metal binding always causes a blue shift in the
absorption spectrum, and in our systems increase the EET ef-
ficiency. In the compound 2b, the energy transfer efficiency was
almost 100%, and increasing the interchromophoric distance should
decrease the EET efficiency to levels appropriate for cation
modulation. Thus, we synthesized new dyes 3-4 shown in Figure
1. Boron to boron distance in dyads 2b, 3b, and 4b is estimated as
11.9, 25.5, and 39.1 Å (Hyperchem version 7.5, PM3 semiempirical
module). For compounds 3b, 3c, 4b, and 4c, the absorbance spectra
very clearly show two prominent peaks in the visible region, as
expected (Supporting Information). The emission spectra of these
compounds are very informative. The changes in the emission
characteristics reflect the changes in the EET efficiency. In
compound 2b, there is very weak emission from the shorter
wavelength emitting dye, but in compounds 3b and 4b, a progres-
sively larger fraction of excitation energy appears as emission at
540 nm (Figure 2). This is also in accordance with the decreased
emission intensity at the longer wavelengths (670 nm). Energy
transfer efficiencies were calculated using quantum yields of
emission (Supporting Information). In order to obtain a cation
responsive chemosensor, we took advantage of our modular design
and switching to a dithiaazacrown-substituted benzaldehyde instead
of 4-dimethylaminobenzaldehyde, we obtained compounds 3c and
4c. The specific dithiazacrown we selected, is reportedly selective
for Hg(II) ions in a range of solvents.6 This is apparent in our
experiments (Figure 3) as well, the emission ratio changes 35-fold
on binding to Hg(II). This is a remarkable value for a ratiometric
fluorescent chemosensor. The emission response of 4c to Hg(II)
ions is also shown in Figure 4. Clearly, Hg(II) binding (Kd in THF
was determined to be 4.5 × 10-7 M, see Supporting Information)
causes a spectral shift in the absorbance of the longer wavelength
dye, increases the spectral overlap between the energy donor and
the acceptor, and an enhanced emission peak is observed. More
efficient energy transfer is corroborated with the changes at the
540 nm.
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J. AM. CHEM. SOC. 2006, 128, 14474-14475
10.1021/ja066144g CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Figure 4. The emission response of the dyad 4c in THF (1.0 µM) to an
increasing concentration (0-25 µM) of Hg(II) ions. A small decrease in
the intensity at 540 nm is indicative of larger EET. The inset shows the
increasing spectral overlap on metal binding: The emission peak (a) of the
compound 4a and absorption peak of metal-bound compound (b) and free
(c) 1 were normalized while keeping the peak ratios between 1-Hg and
free 1 unchanged. The absporption peaks were moved 7 nm to account for
the spectral difference between 1 and 4c.
Figure 2. EET efficiency as a function of interchromophoric distance: as
the distance between the two boradiazaindacene chromophores increases,
the emission peak at 540 nm become more prominent. Concentrations of
the dyes (2b, 3b, and 4b) were set at 1.0 µM, and the excitation was at 500
nm with 5 nm slit widths. The inset shows the boron to boron distance in
Å and the corresponding EET efficiency.
be obtained, if the binding of the analyte modulates the energy
transfer efficiency. We are confident that in due course this general
and modular approach in ratiometric chemosensor design would
lead to novel and useful chemosensors for many biologically and
environmentally relevant analytes.
Acknowledgment. This work was supported by Turkish Sci-
entific and Technological Research Council (TUBITAK) and
Turkish Academy of Sciences (TUBA). A.C. thanks TUBITAK
for a scholarship.
Supporting Information Available: Syntheses, experimental de-
tails, 1H, 13C NMR spectra, and additional spectroscopic data. This
material is available free of charge via the Internet at http://pubs.acs.org.
Figure 3. Emission ratios (I600/I700) for the boradiazaindacene dyad 4c (1.0
µM) on excitation at 500 nm. Hg(II) causes the largest spectral shift, and
hence results in the most efficient energy transfer for the bound state. All
metal ions were at 50 µM concentration. The data were collected in THF
and metals were introduced as perchlorates, except for silver (triflate).
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