Stable and highly fluorescent europium(III) chelates for time-resolved immunoassays

Article abstract of DOI:10.1021/ic400384f

Derivatives of 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6,-bis{[N,N- bis(carboxymethyl)-amino]methyl}pyridine europium(III) (1) bearing one (6) or two (7) additional iminodiacetate coordinating arms have been synthesized. 6 and 7 were significantly more stable than 1 as evidenced by competition experiments with ethylenediaminetetraacetic acid (EDTA) and 1,4,7,10-tetraazacyclododecane- 1,4,7,10-tetraacetic acid (DOTA). While the luminescence quantum yield of 1 remained modest, the other two complexes displayed substantial luminescence efficiency. The introduction of a supplementary iminodiacetate arm in 6 brought important improvements to both the stability and the luminescence properties of the Eu complex. In contrast, although 7 is more luminescent than 1, the introduction of a second iminodiacetate coordinating arm brings no further benefit on the photophysical properties. The most promising results were obtained with the nine-dentate chelate 6 and its Eu complex, which was conjugated to biotin and applied within the frame of a bioaffinity immunoassay of human C-reactive protein.

Full text of DOI:10.1021/ic400384f

Article
pubs.acs.org/IC
Stable and Highly Fluorescent Europium(III) Chelates for Time-
Resolved Immunoassays
Qi Wang,*,† Katia Nchimi Nono,‡ Markku Syrjanpaa,† Loïc J. Charbonniere,‡ Jari Hovinen,†
̀
̈
̈
̈
and Harri Harma*,†
̈
̈
†Laboratory of Biophysics, University of Turku, FI-20014 Turku, Finland
‡Laboratoire d’Ingen
ierie Moleculaire Appliquee l’Analyse, IPHC, UMR 7178 CNRS/UdS, ECPM, Bat
Becquerel, 67087 Strasbourg Cedex, France
́
́
́
a
̀
̂
iment R1N0, 25 rue
ABSTRACT: Derivatives of 4-[2-(4-isothiocyanatophenyl)-
ethynyl]-2,6,-bis{[N,N-bis(carboxymethyl)-amino]methyl}-
pyridine europium(III) (1) bearing one (6) or two (7)
additional iminodiacetate coordinating arms have been
synthesized. 6 and 7 were significantly more stable than 1 as
evidenced by competition experiments with ethylenediamine-
tetraacetic acid (EDTA) and 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid (DOTA). While the luminescence
quantum yield of 1 remained modest, the other two complexes
displayed substantial luminescence efficiency. The introduction
of a supplementary iminodiacetate arm in 6 brought important
improvements to both the stability and the luminescence
properties of the Eu complex. In contrast, although 7 is more
luminescent than 1, the introduction of a second iminodiacetate coordinating arm brings no further benefit on the photophysical
properties. The most promising results were obtained with the nine-dentate chelate 6 and its Eu complex, which was conjugated
to biotin and applied within the frame of a bioaffinity immunoassay of human C-reactive protein.
(carboxymethyl)-amino]methyl}pyridine5 (1a; Figure 1) is one
INTRODUCTION
■
of the most commonly used biomolecule labeling reagents,
since it fulfills almost all of the above-mentioned requirements.
The most serious drawback of 1 as well as other 7-dentate
lanthanide(III) chelates is that they exhibit rather low chelate
stability limiting their use in applications involving treatments
at elevated temperature, low pH, and in the presence of
additional chelating agents such as ethylenediaminetetraacetic
acid (EDTA). The chelate stability has been increased by using
cyclic chelating moieties,6−8 but neutralization of the net charge
decreases the water solubility.
We report here the synthesis of stabilized derivatives of 1.
The synthetic routes are simple, and the chelates are
significantly more stable and luminescent than 1. The
applicability of the biotin conjugated chelates was successfully
demonstrated in a bioaffinity immunoassay of human C-
reactive protein (hCRP). hCRP is a pentameric protein found
in blood with a monomer molar mass of 25 kDa, which is an
important biomarker for inflammation and cardiovascular
disease.
Luminescent lanthanide(III) chelates have several special
properties that make them excellent tools in homogeneous
bioaffinity assays.1−4 Their large Stokes’ shift has a decreasing
effect on scattering phenomena. The long fluorescence decay
after excitation of these molecules allow time-resolved signal
detection, which eliminates completely the background
luminescence originating, for example, from buffer components,
plastics, and biomaterials. The very narrow emission lines allow
the use of effective filters which diminish the background.
Furthermore, since the lanthanide(III) chelates do not suffer
from concentration quenching it is possible to have several
chelates in close proximity enabling multilabeling. This
phenomenon allows also the development of chelates bearing
several light absorbing moieties.
However, the use of stable chelates in bioaffinity assays
demands optimization of the chelate structure. The optimal
luminescent lanthanide chelate must be stable in the presence
of additional chelators even at low pH and high temperature.
The chelate has optimal emission profile, high hydrophilicity,
small size, good biocompatibility, little effect on biomolecules,
and good energy transfer properties. In addition, the chelate
should be feasible for different energy acceptors, and when
possible, the synthesis of the molecule should be simple, cheap,
and scalable.
EXPERIMENTAL PROCEDURES
■
General Procedures. All reagents and solvents used were of
1
reagent grade. The H and 13C NMR spectra were recorded on a
The commercially available europium(III) chelate of 4-[2-(4-
isothiocyanatophenyl)ethynyl]-2,6,-bis{[N,N-bis-
Received: February 13, 2013
Published: July 9, 2013
© 2013 American Chemical Society
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Figure 1. Structures of Eu(III) chelates and their conjugates.
Bruker Avance 600 MHz NMR spectrometer operating at 600.1337
butoxycarbonylmethyl)aminoethyl]amino}acetate11 (319 mg, 0.80
mmol), and potassium carbonate (dry) (524 mg, 3.80 mmol) were
stirred overnight at 55 °C. The solid was removed by filtration. The
solvent was evaporated in vacuo, and the product was purified on a
silica gel column using 20% (v/v) of ethyl acetate in petroleum ether
1
and 150.9179 MHz for H and 13C, respectively. HR mass spectra
were recorded on a Bruker micrOTOF-Q mass spectrometer. UV−
visible absorption spectra were recorded on a Specord 205 (Analytik
Jena) spectrometer. Steady state emission and excitation spectra were
recorded on a Horiba Jobin Yvon Fluorolog 3 spectrometer working
with a continuous 450 W Xe lamp. Detection was performed with a
Hamamatsu R928 photomultiplier. All spectra were corrected for the
instrumental functions. When necessary, a 399 nm cutoff filter was
used to eliminate the second order artifacts. Phosphorescence lifetimes
were measured on the same instrument working in the phosphor-
escence mode, with 50 μs delay time and a 100 ms integration
window.
1
as the eluent. Yield was 562 mg (89%). H NMR (CDCl3): δ 7.69 (s,
1H), 7.59 (s, 1H), 3.94 (s, 4H), 3.41 and 3.40 (2s, 10H), 2.86 (br s,
4H), 1.41 (s, 27H), 1.38 (s, 18H). 13C NMR (CDCl3): δ 170.45,
170.29, 160.60, 134.49, 124.39, 124.26, 81.10, 81.00, 59.65, 59.42,
+
56.01, 55.81, 52.64, 51.88, 28.13, 28.11. HR-MS for C39H66BrN4O10
required 829.3957 and 831.3937, found 829.4050 and 831.4038.
:
Tetra-tert-butyl 2,2′,2″,2′″-(((((4-((4-aminophenyl)ethynyl)-
pyridine-2,6-diyl) bis(methylene))bis((2-(tert-butoxy)-2-
oxoethyl)azanediyl))bis(ethane-2,1-diyl))bis(azanetriyl))-tet-
raacetate (5a). Compound 4a (364 mg, 0.37 mmol) and 4-
ethynylaniline (52 mg, 0.44 mmol) in the mixture of tetrahydrofuran
(THF, 10 mL) and N,N-diisopropylethylamine (DIPEA, 10 mL) was
deaerated with nitrogen for 5 min. Pd(PPh3)2Cl2 (10.4 mg, 0.015
mmol) and CuI (2.9 mg, 0.015 mmol) were added as the catalysts, and
the reaction mixture was stirred overnight under nitrogen at 60 °C.
The solvents were removed in vacuo, and the product was purified on
a silica gel column using a mixture of 20−30% (v/v) of ethyl acetate in
petroleum ether containing 1% (v/v) triethylamine as the eluent. Yield
was 290 mg (76%). 1H NMR (CDCl3, 50 °C): δ 7.44 (s, 2H), 7.30 (d,
J = 8.45 Hz, 2H), 6.61 (d, J = 8.50 Hz, 2H), 3.96 (br s, 4H), 3.43 (s,
8H), 3.40 (br s, 4H), 2.90 (b, 8H), 1.46 (s, 18H), 1.42 (s, 36H). 13C
NMR (CDCl3, 50 °C): δ 170.55, 158.78, 147.56, 133.32, 132.57,
122.66, 114.54, 111.47, 85.87, 80.83, 80.75, 60.07, 56.24, 52.88, 52.47,
28.17, 28.13. HR-MS for C55H87N6O12+: required 1023.6376, found
1023.6308.
Emission decay profiles were fitted to monoexponential and
biexponential functions using the FAST program from Edinburgh
Instrument or with the Datastation software from Jobin Yvon.
Hydrations numbers, q, were obtained using eq 1, were τH2O and τD2O
respectively refer to the measured luminescence decay lifetimes (in
ms) in water and deuterated water, using AEu= 1.2 and aEu= 0.25 for
9
EuIII
q = ALn(1/τ
− 1/τ
− aLn)
H2O
D2O
(1)
Luminescence quantum yields were measured according to
conventional procedures, with diluted solutions (optical density
<0.05), using [Ru(bipy)3]Cl2 in nondegassed water (Φ = 4.0%) as
reference.10 The estimated relative error is 15%.
Compound 2 was synthesized according to literature procedures.5
Tetra-tert-butyl 2,2′,2″,2′″-{{{[(4-bromopyridine-2,6-diyl)bis-
(methylene)]bis([2-(tert-butoxy)-2-oxoethyl]azanediyl}}bis-
(ethane-2,1-diyl)}bis(azanetriyl)}tetraacetate (4a). A mixture of
4-bromo-2,6-bis(bromomethyl)pyridine (2;5 344 mg, 1.0 mmol), tert-
butyl{[bis(tert-butoxycarbonyl methyl) aminoethyl]amino}tris-
(acetate)11 (843 mg, 2.10 mmol) and dry potassium carbonate (1.38
g, 10 mmol) in dry acetonitrile (50 mL) were stirred overnight at 55
°C. The solid was removed by filtration. The solvent was evaporated in
vacuo, and the product was purified on a silica gel column using 1%
(v/v) triethylamine in dichloromethane as the eluent. Yield was 0.56 g
(57%). 1H NMR (CDCl3): δ 7.59 (s, 2H), 3.79 (br, 4H), 3.39 (s, 8H),
3.33 (br, 4H), 2.84 (br, 8H), 1.40 (s, 18H), 1.38 (s, 36H). 13C NMR
(CDCl3): δ 170.48, 160.79, 134.32, 124.09, 80.88, 59.79, 56.05, 53.40,
52.63, 52.06, 28.12, 28.10. HR-MS for C47H81BrN5O12+: required
986.5060 and 988.5040, found 986.4993 and 988.4985.
Di-tert-butyl 2,2′-(((4-((4-aminophenyl)ethynyl)-6-(((2-(bis(2-
(tert-butoxy)-2-oxoethyl)amino)-ethyl)(2-(tert-butoxy)-2-
oxoethyl)amino)methyl)pyridin-2-yl)methyl)azanediyl)-
diacetate (5b). The synthesis was performed as above for compound
5a but using 4b (562 mg, 0.68 mmol) as the starting material. Yield
1
was 421 mg (72%). H NMR (CDCl3): δ 7.45 (s, 1H), 7.35 (s, 1H),
7.18 (d, J = 8.46 Hz, 2H), 6.51 (d, J = 8.52 Hz, 2H), 4.17 (br s, 2H),
3.91 (s, 2H), 3.81 (s, 2H), 3.38 (s, 4H), 3.35 (s, 4H), 3.27 (s, 2H),
2.77 (t, d, J = 6.54, 25.21 Hz, 4H), 1.37 (s, 27H), 1.33 (s, 18H). 13C
NMR (CDCl3): δ 170.68, 170.55, 170.38, 159.06, 158.64, 147.93,
133.18, 133.15, 122.47, 122.38, 114.38, 110.70, 94.80, 85.66, 80.89,
80.72, 80.70, 60.21, 60.08, 59.63, 56.14, 56.09, 55.71, 52.55, 52.16,
28.09, 28.07, 28.04. HR-MS for C47H72N5O10+: required 866.5274,
found 866.5294.
Di-tert-butyl 2,2′-{{{6-{{{2-(bis[2-(tert-butoxy)-2-oxoethyl]-
amino}ethyl}[2-(tert-butoxy)-2-oxoethyl]amino}methyl}-4-bro-
mopyridin-2-yl}methyl}azanediyl}diacetate (4b). Di-tert-butyl
2,2′-{[4-bromo-6-(bromomethyl)pyridin-2-yl]methylenenitrilo}bis-
(acetate) (3)8 (385 mg, 0.76 mmol), tert-butyl{[bis(tert-
Preparation of Complex 6a. Compound 5b (220 mg, 0.215
mmol) was dissolved in TFA (2 mL) and the mixture was stirred in a
water bath at 25 °C for 2 h. All volatiles were removed in vacuo. The
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Article
a
Scheme 1
a
t
t
t
(a). HN(CH2CO2 Bu)2, K2CO3, acetonitrile, 55 °C. (b). HN(CH2CO2 Bu)(CH2)2N(CH2CO2 Bu)2, K2CO3, acetonitrile, 55 °C. (c). 4-
Ethynylaniline, Pd(PPh3)2Cl2, CuI, DIPEA, THF, 60 °C. (d). TFA, r.t., 2 h. (e). EuCl3, pH 7.0. (f). SCCl2, water-CHCl3, pH 7.0 (g) glycine. (h). N-
Biotinyl-3-aminopropylamine.
HR-MS for 1c C37H41EuN8O10S22−: required 486.0798 and
residue was dissolved in water (2 mL), and a EuCl3 solution (0.236
mmol in 0.5 mL water) was added and stirred for 10 min. The pH was
adjusted to 7.0 with triethylamine, and the mixture was stirred for
another 10 min. The pH was adjusted to 9.0 with sat. Na2CO3. The
precipitate formed was removed by centrifugation. The pH of the
solution was adjusted to 7.0 with acetic acid. Acetone (45 mL) was
added, and the mixture was shaken for 1 min. The precipitate was
collected by centrifugation, washed with acetone (50 mL), and dried
with airflow. HR-MS for C27H27EuN5O10−: required 732.0961 and
734.0975, found 732.1024 and 734.1032. The precipitate was dissolved
in water (1 mL). Chloroform (1 mL) and thiophosgene (0.33 mL, 4.3
mmol) were added, and the mixture was stirred vigorously for 5 min.
The pH was monitored and kept at 7.0 with 15% of NaHCO3 solution.
Chloroform was removed followed by addition of acetone (50 mL)
with stirring. The precipitate formed was isolated by centrifugation,
washed with acetone (50 mL) and dried. HR-MS for
C28H25EuN5O10S−: required 774.0526 and 776.0539, found
774.0649 and 776.0694.
Preparation of Complex 7a. The title compound was prepared
as described above for compound 6a but using compound 5a (234 mg,
0.27 mmol) as the starting material. HR-MS for the amino form
C31H33EuN6O122−: required 416.0683 and 417.0690, found 416.0721
and 417.0740. HR-MS for the isothiocyanato form
C32H31EuN6O12S2−: required 437.0465 and 438.0472, found
437.0521 and 438.0512.
487.0805, found 486.0764 and 487.0780.
HR-MS for 6c C41H48EuN9O12S22−: required 536.6037 and
537.6044, found 536.6014 and 537.6023.
HR-MS for 7c C45H55EuN10O14S22−: required 587.1275 and
588.1282, found 587.1251 and 588.1260.
Conditional Stability Constants Determination. In a typical
experiment,12 batches of 2 mL of about 2 × 10−5 M solutions of the
europium complexes in TRIS buffer at pH = 7.4 were mixed with
various quantities of a stock solution of 5 × 10−2 M EDTA (or
1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)) in
the same buffer, so that the ratio of added EDTA per Eu complex
varies from 0 to 450000. For each solution, the emission spectrum was
measured and the decrease in intensity was fitted to eq 213 using the
nonlinear regression analysis of the SPECFIT software:14
[EuL] + EDTA ↔ [Eu(EDTA)] + L with
KcondEDTA
KcondL
[Eu(EDTA)] × [L]
[EDTA] × [EuL]
K =
=
(2)
The conditional stability constant could be determined using values
of KcondEDTA calculated from literature data.15
hCRP Immunoassay. Human C-reactive protein (hCRP) was
obtained from Orion Diagnostica (Espoo, Finland) and monoclonal
anti-CRP antibody 6404 from Medix Biochemica (Kauniainen,
Finland). Nunc C12 low fluor maxi wells (Thermo Scientific,
Roskilde, Denmark) were coated with 150 ng of 6404 antibody in
40 μL of 50 mM phosphate buffer (pH 7.4) for 16 h at 4 °C. The wells
were washed twice using wash buffer from Kaivogen Oy (Turku,
Finland). Final blocking of the well surface was carried out with 200
μL of 0.1% BSA in the phosphate buffer for 2 h at 25 °C. The same
6404 antibody was conjugated with d-biotin NHS ester (Sigma-
Aldrich, St. Louis, U.S.A.) using 20-fold excess in 50 mM phosphate
buffer (pH 7.8). After conjugation the antibody was purified with
NAP-5 column (GE Healthcare, Uppsala, Sweden) using TBS-buffer
(50 mM Tris-Cl pH 7.5, 150 mM NaCl). Three replicates of 0−10
mg/L hCRP were incubated for 60 min in 50 μL of TBS-buffer in
prewashed 6404 coated wells. The wells were washed once, and 50 ng
of biotinylated 6404 antibody was added to the wells in 50 μL of TBS
and incubated for 60 min. The wells were washed once, and 50 ng of
streptavidin (BioSpa, Milan, Italy) in 50 μL of TBS was added,
incubated for 10 min and washed once. Thereafter, 50 μL of 40 nM
biotinylated europium chelates were added and incubated for 10 min.
The wells were washed twice and measured with a Victor2 1420
multilabel counter (PerkinElmer, Wallac OY, Turku, Finland) in time-
resolved luminescence mode using an excitation wavelength of 340 nm
and an emission wavelength of 615 nm, a 400 μs delay, and 400 μs
integration times.
Preparation of Glycine-Complexes 1b, 6b, and 7b. The
isothiocyanates (1a, 6a, 7a; 20 mg each) were allowed to react with
glycine (300 mg) at pH of about 7. The product was purified by
HPLC (column: Supelco Ascentis RP-Amide, 21.2 mm × 25 cm.
Particle 5 μm, flow rate 8.0 mL/min; eluent 20 mM TEAA buffer in
2−25% acetonitrile, v/v). The fractions were collected and
concentrated. The salts were removed on HPLC by using the
above-mentioned system by omitting the buffer component from the
eluent.
HR-MS for 1b C26H22EuN5O10S2−: required 373.5148 and
374.5155, found 373.5163 and 374.5173.
HR-MS for 6b C30H29EuN6O12S2−: required 424.0387 and
425.0393, found 424.0398 and 425.0380.
HR-MS for 7b C34H36EuN7O14S2−: required 474.5625 and
475.5632, found 474.5676 and 475.5690.
Preparation of Biotin-Complexes of 1c, 6c, and 7c. The
isothiocyanates (1a, 6a, 7a; 1 mg each) were allowed to react with N-
Biotinyl-3-aminopropylamine (TFA salt, 5 mg each) at pH of about
8.0. The product was purified by HPLC (column: Supelco Ascentis
RP-Amide, 4.6 mm × 15 cm, particle 5 μm, flow rate 1.0 mL/min;
eluent 20 mM TEAA buffer at pH 7.0 with 2−60% methanol, v/v).
The fractions were collected and dried in vacuum.
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Table 1. Spectroscopic Data for the Eu Complexes in 0.01 M TRIS/HCl Buffer at pH 7.4
compound
λmax/ nm
ε/M−1 cm−1
τH2O /ms
τD2O /ms
q
ΦOv
ηeff
ΦEu
1b
6b
7b
319
317
318
16350
24350
20900
0.39
1.15
1.07
2.10
1.96
1.69
2.2
0.1
0.1
0.06
0.14
0.12
0.54
0.45
0.41
0.11
0.31
0.29
Figure 2. UV−vis absorption (blue), excitation (λem = 615 nm, red) and emission (λexc = 319 nm, green) spectra of 1b in 0.01 M TRIS/HCl buffer
at pH 7.4.
Figure 3. Emission spectra of the 1b, 6b, and 7b complexes (from bottom to top) in TRIS/HCl buffer (0.01 M, pH 7.4; λexc = 318 nm).
Spectroscopic Properties of the Chelates. The
spectroscopic properties of the complexes were measured in
0.01 M TRIS/HCl buffer at pH 7.4 on the glycine
functionalized complexes (1b, 6b, 7b), and the most important
parameters are gathered in Table 1. The UV−vis absorption
spectra of the three Eu complexes are very similar, displaying a
strong absorption band centered at about 318 nm, correspond-
ing to π→π* transitions on the pyridyl rings (see Figure 2 for
1b). The presence of the para-(thiourea)-toluyl substitution
resulted in a strong bathochromic shift of this absorption band,
when compared to nonsubstituted pyridines for which the
maximum of absorption can be found at 265−267 nm.17,18
Upon excitation into the π→π* absorption bands, all
complexes display well resolved emission bands between 575
and 730 nm associated with f-f transitions on the europium
ion.19 These emission bands correspond to the 5D0→7FJ
RESULTS AND DISCUSSION
■
Syntheses of the Chelates. Synthesis of the europium-
(III) chelates is depicted in Scheme 1. Accordingly, treatment
of 4-bromo-2,6-bis(bromomethyl)pyridine (2)5 and di-tert-
butyl 2,2′-(((4-bromo-6-(bromomethyl)pyridin-2-yl)methyl)-
azanediyl)diacetate (3)8 with (tert-butyl{[bis(tert-
butoxycarbonylmethyl)aminoethyl]amino}-acetate,11 gave rise
to 4a,b. They were subsequently converted to the correspond-
ing ethynyl derivatives (5a,b) by Sonogashira coupling reaction
with 4-ethynylaniline. Removal of the tert-butyl groups by
acidolysis followed by treatment with europium(III) chloride
afforded the corresponding europium(III) chelates. Finally,
treatment with thiophosgene16 gave the desired activated
chelates 6a and 7a.
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■
●
▲
Figure 4. hCRP immunoassays with biotin-conjugated europium chelates: 1c ( ), 6c ( ), and 7c ( ). The lower limit of detections for the biotin
chelates were 6.5, 1.5, and 1.9 μg/L, respectively. The excitation wavelength is 340 nm, and the emission wavelength is 615 nm.
transitions with J = 0 (single band at 575 nm), J = 1 (between
578 and 600 nm), J = 2 (strong, 605 to 625 nm), J = 3 (weak
around 650 nm) and J = 4 (690 to 715 nm). The
corresponding excitation spectra are perfectly superimposable
with the absorption spectra, evidencing an efficient ligand to
metal energy transfer process that is a good antenna effect.20
The luminescence decay profiles measured at the maximum of
emission were all perfectly fitted with monoexponential
functions, pointing to the presence of single species in solution.
As expected, the complex obtained from the heptadentate
ligand of 1b displayed the shorter lifetime (0.39 ms), the
coordination sphere of the europium being probably
unsaturated. This was confirmed by the calculation of the
hydration numbers of the complexes according to the method
developed by Horrocks,21 using Beeby’s coefficients9 (Table 1).
While the heptadentate ligand of 1b releases the place for two
inner sphere water molecules, the replacement of one or two
acetate functions by iminodiacetate ones resulted in the
fulfilment of the coordination sphere and the removal of
solvent molecules from the first coordination sphere.
41 to 54%, as expected for a similar antenna unit. In contrast,
the metal centered quantum yield of 1b is largely affected by
the presence of the inner sphere water molecules, losing two-
thirds of the value obtained when the coordination sphere is
saturated. Finally, the introduction of a second iminodiacetate
coordinating arm in 7b has no significant further impact on the
photophysical properties, but it can provide extra stability to the
chelate.
Determination of the Conditional Stability Constants.
The determination of the conditional stability constants of the
different complexes was addressed by means of competition
experiments with EDTA and DOTA. Only in the case of 1b
was it possible to displace the equilibrium in the presence of
EDTA (from 1 to 5000 equiv). Considering a conditional
stability constant of 14.53 Log units for EDTA at this pH,15 the
fitting resulted in a conditional stability constant of 16.72 Log
units for 1b at pH = 7.4. This value is very similar to reported
values in the literature for similar coordination environments
such as in the case of pyridyl chelates with H or OMe functions
in the para position (Log Kcond = 16.47 and 16.67, as calculated
from refs 17 and 18). For both 6b and 7b, even in the presence
of large excess of EDTA (up to 1000 equiv in both cases), it
was not possible to displace the equilibrium, and to extract the
Eu atom from its coordinating ligand. We thus turned our
attention toward DOTA, known to form very stable complexes
with Ln cations,25 as competing ligand, but even after 3 days at
80 °C in the presence of 50 equiv, it was not possible to
observe demetalation. Although this last situation may be due
to kinetic inertness of the complexes, the experiments with
EDTA and DOTA allowed us to estimate a lower limit of 21
Log units for the values of the conditional stability constants of
6b and 7b with europium. It is believed that 7b can be more
stable than 6b although the results are the same in our
experimental conditions.
The europium ion centered emission spectra of the
complexes are presented in Figure 3. As expected for low
symmetry complexes,22 the D0→7F2 transition represents the
5
most intense emission band. The main variations in the series
are observed on this transition and on the pattern of the
5D0→7F4 transition, this transition becoming more intense
(compared those with J = 0 to 3) for 7b.
Based on the emission spectra of the three complexes (Figure
3), it was possible to determine the europium centered
quantum yield, ΦEu, using the methodology developed by
Werts and co-workers,23,24 from which one can calculate the
sensitization efficiency, ηeff, that reflects the capacity of the
ligand to transfer the absorbed energy to the europium, using
eq 3:
Conjugation to Bioactive Molecules and hCRP
Immunoassay. The applicability of the chelates were
demonstrated in a sandwich-type hCRP immunoassay. There-
fore, the chelates 1b, 6b, and 7b were conjugated with N-
Biotinyl-3-aminopropylamine. The synthesized chelates 6c and
7c performed equally well in the hCRP assay (Figure 4). The
ΦOv = η × ΦEu
(3)
eff
Where ΦOv is the overall luminescence quantum yield
measured by the direct method (Table 1).
The calculated values of ηeff and ΦEu evidenced that the
sensitization is almost the same for all compounds ranging from
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Inorganic Chemistry
Article
(17) Pellegatti, L.; Zhang, J.; Drahos, B.; Villette, S.; Suzenet, F.;
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6593.
biotin-chelate 1c resulted in a lower luminescence signal than
the 6c and 7c which is in accordance with the measured
quantum yields and emission lifetimes. The analytical detection
limits for 1c, 6c, and 7c were 6.5, 1.5, and 1.9 μg/L,
respectively, as calculated from 3 SD above the mean of the
zero hCRP concentration and from the following equations, 1c:
y = 32515x − 34, R2 = 0.96; 6c: y = 87730x +16, R2 = 0,99; 7c:
y = 90760x − 31, R2 = 0.99.
In summary, the stability of 4-[2-(4-isothiocyanatophenyl)-
ethynyl]-2,6,-bis{[N,N-bis(carboxymethyl)-amino]methyl}-
pyridine europium(III) can be dramatically enhanced by
addition of an iminodiacetate coordinating arm to the chelate
structure. This modification has also a positive effect on the
luminescence quantum yield. The synthetic protocol is
straightforward and applicable to other chelates bearing various
substituents at 4-position of pyridine moiety.26 The bio-
conjugated chelates were also demonstrated to be highly
suitable for diagnostic assay purposes as the immunoassays
using the biotin labels cover the clinical range of the hCRP
requirements.27
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: qiwang@utu.fi (Q.W.), harri.harma@utu.fi (H.H.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported financially by the Finnish Academy
Research Project.
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