Signal amplification via intramolecular energy transfer using tripodal neutral iridium(III) complexes upon binding to avidin

Article abstract of DOI:10.1021/ja710803p

A neutral tripod system for biotin-avidin assays, which is composed of biotin, FIrpic as an energy acceptor, and mCP as an energy donor, offers remarkable sensitivity over traditional transition metal based protein probes due to the intramolecular energy transfer and increased hydrophobicity associated with the avidin binding site and neutral probe 1. Copyright

Full text of DOI:10.1021/ja710803p

Published on Web 03/04/2008
Signal Amplification via Intramolecular Energy Transfer Using Tripodal
Neutral Iridium(III) Complexes upon Binding to Avidin
Tae-Hyuk Kwon, Jongchul Kwon, and Jong-In Hong*
Department of Chemistry, College of Natural Sciences, Seoul National UniVersity, Seoul 151-747, Korea
Received December 4, 2007; E-mail: jihong@snu.ac.kr
The extremely strong binding interaction between biotin and
(strept)avidin has widespread bioanalytical applications such as the
detection of biomolecules, clinical diagnosis, and immunoassays.1
Of the many techniques for biotin-(strept)avidin assays,2 there has
been considerable interest in transition metal complexes using
phosphorescence to overcome the problems of organic fluorophores
such as the strong pH dependence, low photostability, small Stokes
shifts, and short lifetimes.3-6 Among them, Ir(III) complexes are
Figure 1. Molecular structures of the neutral probes with an energy donor
(1) and without an energy donor (2).
promising candidates in various bioanalytical applications on
account of their intense emission, wide range of emission energies,
and high luminescence quantum yield.5a However, most ionic
transition metal complexes developed as biological labeling reagents
did not show high sensitivity upon binding to (strept)avidin.3-5 For
example, the luminescence enhancement factor of previous transi-
tion metal complexes upon binding to (strept)avidin is usually in
the range of ca. 1-3 fold. This is because the luminescence
enhancement only results from the increased hydrophobicity of the
surrounding environment of the bound probes, compared with the
environment of the free probes.3-5
Figure 2. Luminescence intensity changes for (I) the titration of avidin
This study reports a tripod system (1) for biotin-avidin assays,
which consists of an energy acceptor, an energy donor, and biotin
(Chart 1). This system is expected to provide a dramatic increase
in luminescence intensity due to the intramolecular energy transfer
between the donor and acceptor, as well as the increased hydro-
phobicity of the neutral probe, upon binding to avidin. To the best
of our knowledge, this is the first luminescent biotin-transition
metal complex conjugate using intramolecular energy transfer for
luminescence enhancement. Neutral systems can offer a more
hydrophobic environment than ionic probes. Therefore, they are
expected to show a high binding affinity3 and reduce the nonspecific
interaction7 that can exist between the protein surface and ionic
transition metal probe. Iridium(III) bis[(4,6-difluorophenyl)-pyridi-
nato-N,C2′]picolinate (FIrpic), which is a well-known sky blue
dopant in OLEDs, was selected as an acceptor on account of its
high quantum efficiency, and N,N′-dicarbazolyl-3,5-benzene (mCP)
was chosen as an energy donor that demonstrates higher singlet
and triplet energies than those of the acceptor.8a Control probe 2
without a donor part, which has a similar structure to that of
previous transition metal probes but is electronically neutral, was
also prepared (Figure 1). The luminescence of probes 1 and 2 results
from both 1MLCT (dπ(Ir)fπ*(N-O)) and 3LC in the FIrpic moiety
and has a similar emission spectrum (λmax ) 472 nm) to that of
FIrpic.8b There is a good overlap between the emission spectrum
of the donor (mCP unit) and the absorption spectrum of FIrpic over
(1.66 µM) with 1, (II) 1 in the buffer A solution9 at 310 nm excitation, (V)
the titration of avidin (1.66 µM) with 2, and (VI) 2 in the buffer A solution,
upon excitation at 380 nm.
Chart 1. Schematic Diagram of a Neutral Tripod Probe System
binding to avidin, the neutral tripod 1 showed a dramatic increase
in emission intensity when excited at the donor absorption peak
(310 nm), rather than at the MLCT region (380 nm) of the acceptor
(Chart 1 and Figure 2, Vide infra for details).
The avidin binding properties of probes 1 and 2 were investigated
using the standard HABA (4,4′-hydroxyazobenzene-2-carboxylic
acid) assays,1,3-5 which are based on the competition between biotin
and HABA for binding to avidin, and luminescence titration
experiments. The addition of probe 1 or 2 into a mixture of HABA
(1 mM) and avidin (1.66 µM) results in a decrease in absorbance
at 500 nm.9 This shows that the bound HABA molecules are
replaced by probes 1 and 2 with the same stoichiometry as that of
free biotin (4 (1 or 2):1 (avidin)).
Luminescence titration using probe 113 showed the turning point
at 4 equiv of 1 due to the strong interaction of the biotin moiety9,14
with the four binding sites of avidin (I, Figure 2). Luminescence
titration using probe 1 also shows a remarkable increase in emission
intensity (I) (ca. 14-fold) at 1/avidin ) 4:1, when excited at the
3
350 nm (1MLCT and LC region), which ensures singlet-singlet
energy transfer from mCP to FIrpic.9-11 Furthermore, the distance
between the donor and acceptor (∼15 Å, under the assumption that
all the bonds are linked through a trans geometry) in the optimized
geometric structure of probe 1 and density functional theory (DFT)
calculations suggest that the effective triplet-triplet energy transfer
between the donor and acceptor can also occur.9,10,12 Indeed, upon
9
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J. AM. CHEM. SOC. 2008, 130, 3726-3727
10.1021/ja710803p CCC: $40.75 © 2008 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Photophysical Properties and Lifetime of 1 and 2 at 298
K
fore, this new tripod system can be used effectively for labeling of
biomolecules.
b
medium
λmax/nm (ΦPL
)
τ
(µ
s)c
τ
(µ
s)d
τ (µ
s)e
A homogeneous competitive biotin assay was carried out by
adding free biotin in the range of 10-11 to 10-3 M to a mixture of
probe 1 (6.64 µM) and avidin (1.66 µM) in buffer A solution. After
1 h of incubation, the emission intensity of probe 1 decreased
gradually according to the concentration of free biotin because the
binding of free biotin to avidin (Kd ) ca. 10-15 M) is much stronger
than that of probe 1 (Figure 3). Due to the high emission intensity
of the 1-avidin complex, the linear range for the detection of the
free biotin analyte was increased greatly to 10-6.5-10-10.5 M.
Compared with ionic transition metal probes, the detection limit
of the current system is higher by 2 or 3 orders of magnitude.3-5
In summary, we developed the first phosphorescent sensing
system that can overcome intrinsic limitations in the sensitivity of
the previous biotin-avidin assays. The neutral sensing system for
biotin-avidin assays offers remarkable sensitivity over traditional
transition metal based probes, due to the intramolecular energy
transfer and increased hydrophobicity associated with the avidin
binding site and neutral probe 1, and can be used as a homogeneous
competitive assay for biotin. New biomolecule probe systems can
be produced if biotin is replaced with other recognition elements
for various biomolecules.
1
2
CH2Cl2
CH3CN
H2Oa
CH2Cl2
CH3CN
H2Oa
472, 519 (0.28)
473, 498 (0.14)
474, 505 (0.01)
469, 496 (0.10)
472, 493 (0.03)
463, 501 (0.01)
1.01
0.49
0.28
0.92
0.45
0.26
0.56
0.49
0.28
0.25
a pH 7.5, buffer A solution (H2O/DMSO ) 9:1). bQuantum yield was
c
measured using FIrpic (ΦPL)0.42) as a reference. [1] ) [2] ) 6.64 µM,
[avidin] ) 0 µM. d[1] ) [2] ) 6.64 µM, [avidin] ) 1.66 µM. e[1] ) [2] )
6.64 µM, [avidin] ) 1.66 µM, [biotin] ) 166 µM.
Acknowledgment. We thank the SSDIP and Seoul R&BD for
their financial support. BK 21 fellowship grants were provided to
T.-H.K. and J.K.
Figure 3. Homogeneous competitive assay for biotin using probe 1 (6.64
µM) and avidin (1.66 µM). The emission intensity was measured at 472
nm.
Supporting Information Available: Experimental procedures,
spectral data, UV and PL data, measurement of ET efficiency, and
titration data. This material is available free of charge via the Internet
at http://pubs.acs.org.
donor (mCP unit) absorption peak (310 nm), compared with that
of compound 2 (V) without an energy donor upon 4:1 binding with
avidin when excited at the MLCT region (380 nm) (Figure 2).
Furthermore, the emission intensity of the probe 1-avidin complex
dramatically increases with increasing concentration of probe 1 until
it fully binds to avidin and is ca. 4-fold higher than that of probe
1 itself (II) when excited at 310 nm. At over 4 equiv, the slope of
the probe 1-avidin complex (I) becomes similar to that of probe
1 (II) without avidin. This means that the increase in emission with
more than 4 equiv of probe 1 just reflects the increase in the probe
concentration. Therefore, a nonspecific interaction between the free
probes and the protein surface can be excluded.4b,7 Compound 8
without a biotin moiety9 showed no increase in emission intensity
in the presence of avidin (Figure S9).
In contrast, the luminescence titration with probe 2 shows that
the emission intensity of the probe 2-avidin complex (V) is ca.
1.6 times that of probe 2 in the absence of avidin (VI).9 The
emission intensity of probe 1 (II) excited at 310 nm was significantly
larger than that of probe 2 (VI) excited at 380 nm and increased
with increasing concentration of the probe due to intramolecular
energy transfer. This indicates that intramolecular energy transfer
References
(1) (a) Wilchek, M.; Bayer, E. A. Methods of Enzymology; Academic Press:
San Diego, CA, 1990; Vol. 184. (b) Hermanson, G. T. Bioconjugate
Techniques; Academic Press: San Diego, CA, 1996.
(2) (a) Gorman, E. V.; Rothenberg, J. M.; Baker, E. A.; Wilcheck, M. Methods
Enzymol. 1990, 184, 217. (b) Barbarakis, M. S.; Qaisi, W. G.; Daunert,
S.; Bachas, L. G. Anal. Chem. 1993, 65, 457. (c) Haddour, N.; Cosnier,
S.; Gondran, C.; Chem. Commun. 2004, 2472.
(3) Lo, K. K.-W.; Hui, W.-K.; Chung, C.-K.; Tsang, K. H.-K.; Lee, T. K.-
M.; Li, C.-K.; Lau, J. S.-Y.; Ng, D. C.-M. Coord. Chem. ReV. 2006, 250,
1724.
(4) (a) Lo, K. K.-W.; Hui, W.-K.; Ng, D. C.-M.; Cheung, K.-K. Inorg. Chem.
2002, 41, 40. (b) Lo, K. K.-W.; Hui, W.-K.; Ng, D. C.-M. J. Am. Chem.
Soc. 2002, 124, 9344. (c) Slim, M.; Sleiman, H. F. Bioconjugate Chem.
2004, 15, 949. (d) Lo, K. K.-W.; Tsang, K. H.-K.; Sze, K.-S. Inorg. Chem.
2006, 45, 1714.
(5) (a) Lo, K. K.-W.; Ng, D. C.-M.; Chung, C.-K. Organometallics 2001,
20, 4999. (b) Lo, K. K.-W.; Chung, C.-K.; Ng, D. C.-M.; Zhu, N. New J.
Chem. 2002, 26, 81. (c) Lo, K. K.-W.; Chan, J. S.-W.; Lui, L.-H.; Chung,
C.-K. Organometallics 2004, 23, 3108.
(6) Wong, K. M.-C.; Tang, W.-S.; Chu, B. W.-K.; Zhu, N.; Yam, V. W.-W.
Organometallics, 2004, 23, 3459.
(7) Kim, T. W.; Yoon, H. Y.; Park, J.-h.; Kwon, O.-H.; Jang, D.-J.; Hong,
J.-I. Org. Lett, 2005, 7, 111.
can be an effective method for increasing the sensitivity (ΦET
>
(8) (a) Holmes, R. J.; Forrest, S. R.; Tung, Y.-J.; Kwong, R. C.; Brown, J. J.;
Garon, S.; Thompson, M. E. Appl. Phys. Lett. 2003, 82, 2422. (b)
Lammansky, S.; Djurovich, P.; Murphy, D.; Abdel-Razzaq, F.; Lee, H.-
E.; Adachi, C.; Burrows, P. E.; Forrest, S. R.; Thompson, M. E. J. Am.
Chem. Soc. 2001, 123, 4304.
74%, in avidin + 1).9 In addition, as shown in the Supporting
Information, the emission intensity of probe 1 (IV) in the presence
of avidin was much lower when excited at 380 nm (MLCT region
of FIrpic) compared to 310 nm (I).9 The lifetime of probes 1 and
2 in the presence of avidin was elongated ca. 2.0- and 1.9-fold,
respectively, due to the hydrophobic environment of the biotin
binding sites of avidin (Table 1). This was supported by the fact
that the lifetimes of probes 1 and 2 decrease with increasing solvent
polarity (Table 1). The addition of excess biotin to a 1:4 mixture
of avidin and probe 1 (or 2) restored the original lifetime of probe
1 (or 2) before complexation. Therefore, the increase in emission
intensity results from intramolecular energy transfer and the
hydrophobicity associated with the binding sites of avidin. There-
(9) See the Supporting Information.
(10) Kwon, T.-H.; Kim, M. K.; Kwon, J.; Shin, D.-Y.; Park, S. J.; Lee, C.-L.;
Kim, J.-J.; Hong, J.-I. Chem. Mater. 2007, 19, 3673.
(11) The singlet-singlet intramolecular energy transfer in 1 from mCP to FIrpic
exhibits a high efficiency of 92% via the transient PL method.9,10
(12) The triplet-triplet energy transfer efficiency of 1 was estimated to be
99% via the transient PL method.9,10
(13) Due to the solubility problem of probes 1 and 2 in aqueous solvent, H2O/
DMSO (9:1, v/v) was used in all the titrations.
(14) Wang, Z.-X.; Kumar, N.R.; Srivastava, D. K. Anal. Biochem. 1992, 206,
376.
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