A near-infrared luminescent label based on YbIII ions and its application in a fluoroimmunoassay

Article abstract of DOI:10.1002/1521-3773(20001215)39:24<4542::AID-ANIE4542>3.0.CO;2-C

The combination of the long-lived, spectrally pure emission of Yb^III ions with the outstanding light absorbing capabilities of organic dyes gives photo luminescent lanthanide complexes that are interesting candidates for use as a new class of luminescent labels for molecules of biomedical interest. The water-soluble complex shown can be covalently attached to biomolecules and used in a model medical diagnostic test.

Full text of DOI:10.1002/1521-3773(20001215)39:24<4542::AID-ANIE4542>3.0.CO;2-C

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[12] Several solvents were tested and noncomplexing solvents, such as
dichloromethane or 1,2-dichloroethane, were optimal.
[13] E. K. Lehnert, J. S. Sawyer, T. L. Macdonald, Tetrahedron Lett. 1989,
30, 5215 ± 5218.
Research on luminescent lanthanide complexes has been
almost exclusively devoted to europiumCiii) and terbiumCiii)
complexes.[6±8] The energetics of sensitized luminescence,
however, dictate that those complexes must be excited with
ultraviolet light,[9] which is not desirable when working with
vital biosystems, requires special optics, and causes extensive
scattering in inhomogeneous media. Although some recent
work has demonstrated that the excitation window for EuIII
complexes can be extended to the violet edge of the visible
region[10] or even to the blue region,[11] it would be desirable to
use significantly longer wavelengths for excitation. Lantha-
nide ions that emit in the near-infrared, such as ytterbiu-
mCiii),[12] neodymiumCiii),[13] and erbiumCiii), may be excited
through organic dyes that absorb at wavelengths extending
towards the red region of the spectrum. With their potential
use as luminescent labels in mind we designed and studied
well-defined, water-soluble near-infrared luminescent lantha-
nide complexes containing organic dyes as the light-absorbing
unit.[14] Recently, we found that 4',5'-bis[N,N-bisCcarboxyme-
thyl)aminomethyl]-fluorescein Cfluorexon, Fx) is a very
efficient sensitizer and a strongly binding ligand for near-
infrared-luminescent lanthanide ions.[15] The thermodynamic
stability of the [YbCFx)] complex is comparable to that of
[YbCEDTA)] CEDTA ˆ ethylenediaminetetraacetate).
[14] Preliminary results indicate that the use of chiral phenols is possible.
Enantioselectivities of the reaction involving these chiral reagents are
encouraging. For example, the reagent derived from the monobenzyl
ether of 3,3'-dimethylbinaphthol converted the benzyl ether of
cinnamyl alcohol into the corresponding cyclopropane with 33% ee.
[15] Katsuki and co-workers have recently reported the use of a 3,3'-
disubstituted binaphthol to perform the enantioselective cyclopropa-
nation of allylic alcohols but six equivalents of Et2Zn were used per
equivalent of phenol Cthreefold excess). Free EtZnCH2I is presumably
formed in this system Cand not ArOZnCH2I): H. Kitajima, K. Ito, Y.
Aoki, T. Katsuki, Bull. Chem. Soc. Jpn. 1997, 70, 207± 217.
A Near-Infrared Luminescent Label Based on
YbIII Ions and Its Application in a
Fluoroimmunoassay**
Martinus H. V. Werts, Richard H. Woudenberg,
Peter G. Emmerink, Rob van Gassel,
Johannes W. Hofstraat, and Jan W. Verhoeven*
Here we report on the ligand FxITC C5; Scheme 1), which is
structurally similar to Fx but carries an isothiocyanate group
CITC) that is reactive towards amine groups and so can be
coupled to proteins. The iminodiacetic acid groups ensure
firm complexation of lanthanide ions Clike they do in
[YbCFx)]), and the dichlorofluorescein chromophore acts as
a sensitizer to near-infrared lanthanide luminescence.
The synthesis of FxITC CScheme 1) reflects the idea of
combining the syntheses of Fx[16] and fluorescein isothiocya-
nate.[17] 2',7'-Dichloro-4-nitrofluorescein diacetate was ob-
tained by condensing 4-nitrophthalic acid with 4-chlororesor-
cinol at 2408C and was separated from the 5-nitro isomer by
fractional crystallization from acetic acid.[18] The dichloro
derivative was chosen to prevent formation of isomers during
the introduction of the methyleneiminodiacetic acid groups.
Moreover, the ªheavyº chlorine atoms might enhance inter-
system crossing in the chromophore which is favorable for
sensitizing lanthanide luminescence.[15]
Direct introduction of the methyleneiminodiacetic acid
groups, as used in the production of Fx,[16] was not successful.
Reaction of iminodiacetic acid dimethyl ester[19] with 2[20] and
subsequent hydrolysis of the ester yielded 3, the desired nitro
precursor to FxITC. Reduction of 3 using H2/Raney Ni
proceded cleanly, but gave rise to the dinickel complex of 4,
which can be observed with electrospray mass spectrometry.
Therefore, 3 was reduced using Na2S/NaHS in dilute NaOH.
After purification by preparative HPLC CC8 column, eluent:
water:acetonitrile:formic acid C155:45:1)), 4 was converted
into FxITC using thiophosgene in acetone.
There has been a great deal of interest in luminescent
lanthanide complexes since Weissmanꢁs discovery that in
these complexes ªthe excitation may be accomplished, under
suitable conditions, through light absorption by other con-
stituents of the rare earth compound with subsequent transfer
of energy to the rare earth ionº.[1] The energy transfer from
organic chromophores to lanthanide ions that he described
provides an effective way to excite the long-lived, sharply
spiked emission. Direct excitation of lanthanide ions is
difficult because of the forbidden nature of the electronic
transitions in these ions. One of the most important applica-
tions of lanthanide complexes is as luminescent labels in
clinical diagnostics where they provide an alternative to
radioactive probes.[2±5] Since lanthanide luminescence decays
much more slowly than the autofluorescence from the bio-
logical material, the latter is suppressed when using time-
gated detection.
[*] Prof. Dr. J. W. Verhoeven, M. H. V. Werts
Laboratory of Organic Chemistry
University of Amsterdam
Nieuwe Achtergracht 129, 1018 WS Amsterdam CThe Netherlands)
Fax : C‡31)20-525-5670
E-mail: jwv@org.chem.uva.nl
Dr. R. H. Woudenberg, P. G. Emmerink, R. van Gassel
Akzo Nobel Central Research
P.O. Box 2300, 6800 SB Arnhem CThe Netherlands)
Prof. Dr. J. W. Hofstraat
Philips Research, Prof. Holstlaan 4
5656 AA Eindhoven CThe Netherlands)
The isopropylamine adduct 6 shows the same complexation
behavior towards lanthanide ions as Fx does.[15] Fluorimetric
titrations with YbIII ions indicate formation of a 1:1 complex
as long as there is no excess YbIII ions. Excess lanthanide ions
leads to the formation of poorly emitting aggregates that so
far have escaped further characterization. Such aggregates,
[**] This work was supported by Akzo Nobel Central Research CArnhem,
The Netherlands). Dr. Fokke Venema and Dr. Harrie Kreuwel of
Organon Teknika B.V. CBoxtel, The Netherlands) are gratefully
acknowledged for discussions and suggestions regarding the diagnos-
tic test, and for supplying the immunochemicals.
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that addition of excess lanthanide ions does not influence the
1:1 antenna:lanthanide stoichiometry in the complex.
The complexes and the avidin conjugates have appreciable
extinction coefficients CTable 1). Their quantum yields and
luminescence lifetimes also compare well to those of
[YbCFx)]. The absolute quantum yields, however, are low.
Table 1. Photophysical data for iPr-FxITC, its YbIII complex, and the protein
conjugates of [YbCFxITC)] in aqueous buffer.
[a]
[b]
Frel emax [mÀ1 cmÀ1
]
lmax [nm][c] tNIR [ms][d]
[YbCFx)][15]
iPr-FxITC
[YbCiPr-FxITC)]
avidin ± [YbCFxITC)] C3.2 l/p) 1.0
avidin-[YbCFxITC)] C5.2 l/p)
anti-hCG ± [YbCFxITC)]
1[e]
±
0.6
125000
125000
145000
502
509
516
514
514
1.9
±
1.8
2.0
2.0
2.2
[f]
[f]
0.8
[a] Frel ˆ near-infrared luminescence quantum yield relative to that of
[YbCFx)]. [b] emax ˆ molar extinction coefficient. [c] lmax ˆ absorption maxi-
mum. [d] tNIR ˆ near-infrared luminescence lifetime monitored at 980 nm.
[15]
[e] The absolute quantum yield of [YbCFx)] in Tris is 7 Â 10À4
.
[f] l/p means
labels per protein.
Scheme 1. Synthesis of FxITC. Reagents and conditions: a) paraformal-
dehyde C90 equiv), 33% HBr in acetic acid, 1108C, 2 h; acetic anhydride
C100 equiv) added dropwise at 208C, reflux, 1 h, 81%; b) iminodiacetic acid
dimethyl ester C15 equiv) in dichloromethane, 90 h at 208C, 69%;
c) methanol/50% NaOH, 16 h at 208C, 99%; d) Na2S, NaHS in 0.6m
NaOH, reflux 15 min; e) thiophosgene C200 equiv) in acetone/water,
30 min at 208C; f) isopropylamine, 8 h at 208C.
This is caused by radiationless deactivation of excited
À
lanthanide ions by molecular vibrations Cespecially C H
[21]
À
and O H oscillators) in the surrounding matrix, a process
which is especially effective with near-infrared-luminescent
lanthanide ions.[22±25] Very recently, Hasegawa et al. succeeded
in pushing the luminescence quantum yield of NdIII ions in
organic complexes up as high as 0.03 by careful design of the
ligands,[26] but such Crelatively) high quantum yields have thus
far only been achieved in organic solvents. In this respect, it
however, are not formed when FxITC is bound to avidin.
Titrating these protein conjugates with YbIII ions yields a
curve typical for the exclusive formation of the 1:1 complex.
Excitation of the avidin conjugate of the YbIII complex
leads to a metal-centered near-infrared luminescence with a
quantum yield equal to that of [YbCFx)]. The corresponding
excitation spectrum closely matches the absorption spectrum
of the complex CFigure 1). No quenching of the luminescence
À
should be noted that closely diffusing O H oscillators Csuch as
water) form a particularly important contribution to the
nonradiative deactivation of the luminescent state of YbIII
complexes.[27]
Although the luminescence quantum yield F is an impor-
tant parameter it is surely not the only one determining the
detectability of a luminescent label. The molar extinction
coefficient e of the label is equally important, and when these
two are combined it is apparent that [YbCFxITC)] performs
quite well in comparison to, for example, the EuIII cryptate
that is used in a commercial diagnostic test.[2] The product eF
for the cryptate in water amounts to approximately
100mÀ1 cmÀ1 when light from a N2 laser with a wavelength of
337nm is used for excitation. [28] The avidin conjugate of
[YbCFxITC)] achieves the same eF value for light with a
wavelength of 514 nm.
The robustness of the label was tested in an archetypal
medical diagnostic test. The format chosen was that of a
simple heterogeneous noncompetitive immunoassay,[5] in
which the analyte to be detected was ªsandwichedº between
an immobilized and a labeled antibody. Antibodies to hCG
Canti-hCG 147b) were immobilized on a small region Cdiam-
eter 3 mm) of a nitrocellulose membrane by impregnation of
0.5 mL of a 2.7 gLÀ1 solution. After drying the membrane, a
mixture of solutions of the [YbCFxITC)]-labeled antibodies
Canti-hCG 293a, 2.7gL À1) and of the sample to be assayed for
hCG Ctotal 20 mL) was spotted onto a different place on the
membrane. This spot was eluted with buffer C100 mL) so that
Figure 1. Luminescence spectra of [YbCFxITC)]-labeled avidin in saline
Tris-HCl buffer CpH 8.3). Left: excitation spectrum Clem ˆ 980 nm). The
visible absorption profile is shown as a dotted line. Right: emission
spectrum Clexc ˆ 510 nm).
by dissolved molecular oxygen was observed, which demon-
strates that the transfer of excitation energy from the organic
chromophore to the lanthanide ion is a rapid and efficient
process. These observations show that the photoluminescence
of the label is not adversely affected by conjugation to a
protein, which furthermore has the desirable consequence
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817L) coupled to a lock-in amplifier CStanford Research SRS530) after
passing it through the dichroic mirror and optical filters to remove the
scattered excitation light. The sample was moved with respect to the
excitation/detection spot using a computer-controlled stage.
eventually the labeled antibodies and the sample passed the
immobilized anti-hCG 147b. The experiment was done both
with a positive sample containing 5000 mIU of hCG plus
labeled antibodies and a blank that only contained the labeled
antibodies. The traces generated by a home-built scanning
near-infrared luminescence microscope clearly showed that a
luminescence signal is generated in the case of a ªpositiveº
test result CFigure 2).
Received: May 26, 2000 [Z15179]
[1] S. I. Weissman, J. Chem. Phys. 1942, 10, 214 ± 217.
[2] G. Mathis, Clin. Chem. 1993, 39, 1953 ± 1959.
[3] A. Mayer, S. Neuenhofer, Angew. Chem. 1994, 106, 1097± 1126;
Angew. Chem. Int. Ed. Engl. 1994, 33, 1044 ± 1072.
[4] I. Hemmilä, J. Alloys Compd. 1995, 225, 480 ± 485.
[5] E. F. Gudgin Dickson, A. Pollak, E. P. Diamandis, J. Photochem.
Photobiol. B 1995, 27, 3 ± 19.
[6] D. Parker, J. A. G. Williams, J. Chem. Soc. Dalton Trans. 1996, 3613 ±
3628.
[7] M. Elbanowski, B. Makowska, J. Photochem. Photobiol. A 1996, 99,
85 ± 92.
[8] N. Sabbatini, M. Guardigli, I. Manet, Adv. Photochem. 1997, 23, 213 ±
278.
[9] F. J. Steemers, W. Verboom, D. N. Reinhoudt, E. B. Van der Tol, J. W.
Verhoeven, J. Am. Chem. Soc. 1995, 117, 9408 ± 9414.
[10] L. J. Govenlock, C. E. Mathieu, C. L. Maupin, D. Parker, J. P. Riehl,
G. Siligardi, J. A. G. Williams, Chem. Commun. 1999, 1699 ± 1700.
[11] M. H. V. Werts, M. A. Duin, J. W. Hofstraat, J. W. Verhoeven, Chem.
Commun. 1999, 799 ± 800.
[12] M. I. Gaiduk, V. V. Grigoryants, A. F. Mironov, V. D. Rumyantseva,
V. I. Chissov, G. M. Sukhin, J. Photochem. Photobiol. B 1990, 7, 15 ±
20.
Figure 2. One-dimensional near-infrared fluorescence microscopy scan of
the region of the nitrocellulose membrane where anti-hCG is immobilized.
The solid line is the near-infrared luminescence signal generated after
elution of a solution of labeled anti-hCG and hCG. The dotted line is the
signal generated by a solution containing only the labeled anti-hCG.
It has been shown that the protein conjugates of [Yb-
CFxITC)] retain photophysical characteristics that are similar
to [YbCFx)], and that they can be applied in a model medical
diagnostic test. Their luminescence is long-lived Cmicrosec-
onds) compared to the optical interferences encountered in
biological matrices, such as fluorescence and scatter, and
resides in the near-infrared region where biological material is
essentially transparent. Most CCD detectors can readily
detect the luminescence of NdIII ions at 880 nm and that of
YbIII ions at 980 nm. These factors, together with the
possibility of exciting these labels with visible light, make
them an attractive new class of luminescent labels. However,
they would benefit from an improvement of their overall
luminescence quantum yield by the suppression[24] of the
nonradiative deactivation[27] of their luminescent states.
[13] S. B. Meshkova, N. V. Rusakova, D. V. Bolshoi, Acta Chim. Hung.
1992, 129, 317± 323.
[14] M. H. V. Werts, J. W. Hofstraat, F. A. J. Geurts, J. W. Verhoeven,
Chem. Phys. Lett. 1997, 276, 196 ± 201.
[15] M. H. V. Werts, J. W. Verhoeven, J. W. Hofstraat, J. Chem. Soc. Perkin
Trans. 2 2000, 433 ± 439.
[16] D. F. H. Wallach, D. M. Surgenor, J. Soderberg, E. Delano, Anal.
Chem. 1959, 31, 456 ± 460.
[17] J. L. Riggs, R. J. Seiwald, J. H. Burckhalter, C. M. Downs, T. G.
Metcalf, Am. J. Pathology 1958, 34, 1081 ± 1097.
[18] R. P. Haugland, J. J. Naleway, Y. Zhang CMolecular Probes),
US 5208148, 1993 [Chem. Abstr. 1993, 119, 112253s].
[19] V. P. Varlamov, S. A. Lopatin, G. E. Bannikova, I. A. Andrushina,
S. V. Rogozhin, J. Chromatogr. 1986, 364, 215 ± 223.
[20] A. W. van der Made, R. H. van der Made, J. Org. Chem. 1993, 58,
1262 ± 1263.
[21] G. Stein, E. Würzberg, J. Chem. Phys. 1974, 62, 208 ± 213.
[22] A. Beeby, S. Faulkner, Chem. Phys. Lett. 1997, 266, 116 ± 122.
[23] A. Beeby, R. S. Dickins, S. Faulkner, D. Parker, J. A. G. Williams,
Chem. Commun. 1997, 1401 ± 1402.
[24] Y. Hasegawa, Y. Kimura, K. Murakoshi, Y. Wada, J.-H. Kim, N.
Nakashima, T. Yamanaka, S. Yanagida, J. Phys. Chem. 1996, 100,
10201 ± 10205.
[25] M. P. Oude Wolbers, F. C. J. M. van Veggel, B. H. M. Snellink-Ruel,
J. W. Hofstraat, F. A. J. Geurts, D. N. Reinhoudt, J. Chem. Soc. Perkin
Trans. 2 1998, 2141 ± 2150.
Experimental Section
Conjugation to avidin: A 2 gLÀ1 solution of FxITC in dry DMSO C40 mL or
80 mL) was added to a 2 gLÀ1 solution of avidin C250 mL, Sigma) in 0.1m
carbonate buffer CpH 9.5). The mixture was incubated for 3 h at room
temperature, with shaking. The solution was diluted to 2.5 mL with a saline
0.05m Tris-HCl buffer CpH 8.3, 0.15m NaCl; Tris ˆ trisChydroxymethyl)-
aminomethane) and purified by gel chromatography on a PD-10 column
CAmersham Pharmacia Biotech) using the saline Tris buffer as an eluent.
The number of labels per protein was determined by means of UV/Vis
spectrometry using the extinction coefficients of the species at 280 and
514 nm.
[26] Y. Hasegawa, T. Ohkubo, K. Sogabe, Y. Kawamura, Y. Wada, N.
Nakashima, S. Yanagida, Angew. Chem. 2000, 112, 365 ± 368; Angew.
Chem. Int. Ed. 2000, 39, 357± 360.
[27] A. Beeby, I. M. Clarkson, R. S. Dickins, S. Faulkner, D. Parker, L.
Royle, A. S. Sousa, J. A. G. Williams, M. Woods, J. Chem. Soc. Perkin
Trans. 2 1999, 493 ± 503.
Optical spectroscopy: The equipment and experimental procedures have
been described in detail previously.[15] Fluorimetry was carried out using a
modified PTI Alphascan apparatus with a Ge detector for the near-infrared
and using a modified Spex Fluorolog 3 with a CCD detector for the near-
infrared measurements.
[28] B. Alpha, R. Ballardini, V. Balzani, J.-M. Lehn, S. Perathoner, N.
Sabbatini, Photochem. Photobiol. 1990, 52, 299 ± 306.
‡
Fluorescence microscopy: The excitation light from an Ar laser C488 nm)
was modulated using a mechanical chopper C40 Hz) and delivered to the
sample via a dichroic mirror and the microscope objective. The emitted
light was detected through the objective by a Ge detector CNorth Coast
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