A europium chelate for quantitative point-of-care immunoassays using direct surface measurement

Article abstract of DOI:10.1021/ac0340051

New labels and assay techniques are needed to improve the sensitivity and quantitativeness of point-of-care immunotesting while sustaining the rapidity and ease of use of the assays. We synthesized a novel, intrinsically fluorescent nonadentate europium c

Full text of DOI:10.1021/ac0340051

Anal. Chem. 2003, 75, 3193-3201
A Europium Chelate for Quantitative Point-of-Care
Immunoassays Using Direct Surface Measurement
Piia von Lode,*,† Jaana Rosenberg,† Kim Pettersson,† and Harri Takalo‡
Department of Biotechnology, University of Turku, Tykisto¨katu 6A, 6th floor, FIN-20520 Turku, Finland, and Innotrac
Diagnostics Oy, Kalevantie 25, FIN-20520 Turku, Finland
instrumentation, as well as the capability of using whole blood as
sample material.
New labels and assay techniques are needed to improve
the sensitivity and quantitativeness of point-of-care im-
munotesting while sustaining the rapidity and ease of use
of the assays. We synthesized a novel, intrinsically fluo-
rescent nonadentate europium chelate with two chro-
mophores and hydrophilic r-galactose side groups. The
chelate is highly fluorescent, soluble in water, and pro-
vides effective shielding of Eu from water. The perfor-
mance of the nonadentate chelate was compared with a
heptadentate chelate in a dry reagent immunoassay for
human chorionic gonadotropin (hCG). After 1 5 -min in-
cubation and washing, time-resolved fluorescence was
measured directly from a wet or dried well surface.
Contrary to the heptadentate label, the effect of aqueous
quenching on the nonadentate label was found to be
insignificant, with calculated analytical detection limits
(background + 3 SD) of 0 .9 and 0 .7 IU/ L hCG for wet
and dry measurements, respectively, and a linear range
up to 5 0 0 0 IU/ L. The CVs for the new label were <8 % at
the cutoff of 2 5 IU/ L and above in both whole blood and
plasma. The novel nonadentate label facilitates short
turnaround times and simple instrumentation due to the
absence of all signal development steps, at the same time
retaining an excellent immunoassay performance.
Currently, many research groups concentrate on the develop-
ment of miniaturized, rapid assay formats based on optical
immunosensors4-8 and microparticles.9,10 These types of assays
facilitate smaller sample volumes and reduced consumption of
reagents and, in some cases, also real-time or multianalyte
measurements. The major challenge especially for sensor systems
intended for clinical use is, however, the discrimination between
specific and nonspecific signals in complex biological matrixes
such as whole blood. Accordingly, only one commercial immun-
osensor application11 has been introduced for point-of-care testing
(POCT) thus far.
Recently, however, several commercial POC immunoanalyzers
have been brought to the market that are based on assay and
detection technologies very similar to those used in advanced
analytical systems.12-14 Although the detection limits are not as
low as those obtained in centralized testing, these pioneering
assays have already shown significantly improved precision of
results when compared to immunochromatographic assay formats,
as well as linear assay ranges exceeding 3 orders of magnitude.
The turnaround times of 15-20 min and the somewhat downsized
instrumentation have been accomplished by reducing the sample
capacity of the analyzers as well as the number and duration of
assay steps. Further simplification of the assay procedures and
the analyzers is, however, still feasible as the current assays
require either on-board centrifugation of whole blood samples,12
separate steps for enzymatic signal enhancement,12,13 or at least
drying of the solid phase prior to direct surface measurement.14
All these extra steps, in addition to being possible sources of error,
In recent years, an ever-increasing number of new point-of-
care (POC) tests have been introduced to the market, most based
on simple manual assay devices using the lateral flow immuno-
chromatographic assay principle. The analytical performances of
most of these assays, however, are not comparable with those of
the complex and sophisticated assay techniques employed in
routine testing. While the most sensitive and reproducible im-
munoassays used in clinical laboratories are based on heteroge-
neous, noncompetitive assay principles,1-3 the design of fully
quantitative, wide-range POC assays with low detection limits
obviously presents great challenges due to the additional demands
for short turnaround times, small size, and ease of use of the
(4) Rowe, C. A.; Scruggs, S. B.; Feldstein, M. J.; Golden, J. P.; Ligler, F. S.
Anal. Chem. 1 9 9 9 , 71, 433-9.
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J. N.; Reichert, W. M. Anal. Chem. 1 9 9 9 , 71, 4344-52.
(6) Schult, K.; Katerkamp, A.; Trau, D.; Grawe, F.; Cammann, K.; Meusel, M.
Anal. Chem. 1 9 9 9 , 71, 5430-5.
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(8) Schneider, B. H.; Dickinson, E. L.; Vach, M. D.; Hoijer, J. V.; Howard, L. V.
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(9) Tarkkinen, P.; Palenius, T.; Lo¨ vgren, T. Clin. Chem. 2 0 0 2 , 48, 269-77.
(10) von Lode, P.; Hagre´n, V.; Palenius, T.; Lo¨ vgren, T. Clin. Biochem. 2 0 0 3 ,
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C. W. Clin. Chem. 1 9 9 9 , 45, 1789-96.
* Corresponding author. Tel: +358-2-333 8060. Fax: +358-2-333 8050.
E-mail: piia.vonlode@utu.fi.
† University of Turku.
‡ Innotrac Diagnostics Oy.
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Messeri, G. Clin. Chem. Lab. Med. 2 0 0 0 , 38, 251-60.
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10.1021/ac0340051 CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/28/2003
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3193

tend to increase the assay times as well as the complexity and,
therefore, the cost and the difficulty of maintenance of the
instrumentation.
In the search for sensitive, fully quantitative yet robust assay
formats for POCT, selection of the label and the detection method
is obviously in a key position. To obtain high sensitivity and assay
precision, a label with a high specific activity and low nonspecific
binding characteristics is required. Time-resolved fluorometry,15-18
regarded as one of the most sensitive detection methods that
currently exist, is one of the very few technologies that can offer
these characteristics and at the same time omit separate signal
enhancement or amplification steps through direct surface mea-
surement of the label. Accordingly, a number of intrinsically
fluorescent lanthanide chelates have been developed and used in
modern bioanalytical applications.14,19-21 A common drawback
affecting the measurement is, however, quenching by water since
particularly europium has a strong tendency to fill its first
coordination sphere with water molecules up to the coordination
number nine. Drying the surface prior to measurement prevents
the nonradiative energy transfer from the europium ions to the
quencher molecules but again results in an increased number of
steps in the assay procedure.14,20,21 The environmental sensitivity
of the fluorophores can, however, be decreased by structural
modifications that exclude water molecules from the coordination
sphere although only few bioanalytical applications using these
kinds of low molecular weight lanthanide chelates22 and cryptates23
have been described.
In this study, we have developed a new intrinsically fluorescent
europiumchelate,{2,2′,2′′,2′′′-{[2-(4-isothiocyanatophenyl)ethylimino]-
bis(methylene)bis{4-{[4-(R-galactopyranoxy)phenyl]ethynyl}-
pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium-
(III) (1 in Figure 1), here referred to as the nonadentate label,
that forms a nine-coordinate complex with the metal ion, thus
providing high thermodynamic stability and minimal fluorescence
quenching by water. Due to the two sugar side groups incorpo-
rated with the chelate structure, the complex is soluble in water
which, for its part, facilitates a gentle and efficient coupling to
antibodies via the aromatic isothiocyanato group of the chelate
and decreases the tendency of nonspecific binding.
Figure 1. Structures of the nonadentate (1) and heptadentate (2)
europiumchelates: {2,2′,2′′,2′′′-{[2-(4-Isothiocyanatophenyl)ethylimino]-
bis(methylene)bis{4-{[4-(R-galactopyranoxy)phenyl]ethynyl}-
pyridine-6,2-diyl}bis(methylenenitrilo)}tetrakis(acetato)}europium(III)
(1); {2,2′,2′′,2′′′-{[4-[(4-Isothiocyanatophenyl)ethynyl]pyridine-2,6-diyl]-
bis(methylenenitrilo)}tetrakis(acetato)}europium(III) (2).
bis(methylenenitrilo)}tetrakis(acetato)}europium(III)25 (2 in Fig-
ure 1), the direct measurement of which has successfully been
employed in immunohistochemistry,20,26 in situ hybridization,26 and
in vitro diagnostics using either microparticles as solid-phase9,10
or a well-based assay format similar to the one described
here.14,27,28 We laid especially high emphasis on studying the
fluorescence characteristics of the chelates in different moisture
conditions, as the aim of the study was to develop a sensitive,
fully quantitative POC assay format with an absolute minimum
number of assay steps, including the direct use of whole blood
samples and no drying of the solid phase prior to measurement.
EXPERIMENTAL SECTION
Materials. The low-fluorescence Maxisorp single wells made
of irradiated polystyrene were purchased from Nunc. The DELFIA
reagents, the heptadentate europium chelate 2 ,25 and the bio-
tinylation reagent biotin isothiocyanate (BITC) were obtained from
PerkinElmer Life Sciences/ Wallac Oy. The monoclonal anti-hCGâ
capture and detection antibodies were acquired from Medix
Biochemica and OEM, respectively, and the streptavidin was from
BioSpa.
We have employed the new label in an all-in-one time-resolved
fluorometric POC assay for human chorionic gonadotropin
(hCG),24 a glycoprotein hormone normally produced by the
trophoblast cells of placenta and used for the early detection of
pregnancy. The performance characteristics of the new label were
compared with those of a well-characterized heptadentate label,
{2,2′,2′′,2′′′-{[4-[(4-isothiocyanatophenyl)ethynyl]pyridine-2,6-diyl]-
Except for the tetra(tert-butyl)2,2′,2′′,2′′′-{[2-(4-aminophenyl)-
ethylimino]bis(methylene)bis(4-bromopyridine-6,2-diyl)bis(meth-
ylenenitrilo)tetrakis(acetate) (6 ), which was synthesized as de-
scribed elsewhere,29 all reagents and solvents used in the organic
syntheses were commercially available and at least of reagent
grade.
(15) Ekins, R. P.; Dakubu, S. Pure Appl. Chem. 1 9 8 5 , 57, 473-82.
(16) Soini, E.; Lo¨ vgren, T. CRC Crit. Rev. Anal. Chem 1 9 8 7 , 18, 105-54.
(17) Hemmila¨, I. Scand. J. Clin. Lab. Invest. 1 9 8 8 , 48, 389-99.
(18) Lo¨ vgren, T.; Pettersson, K. In Luminescence immunoassay and molecular
applications; Van Dyke, K., Van Dyke, R., Eds.; CRC Press: Boca Raton
FL, 1990; pp 233-53.
HCG Calibrators and Clinical Samples. The hCG calibration
solutions were prepared by diluting the Fourth International
(19) Yuan, J.; Matsumoto, K.; Kimura, H. Anal. Chem. 1 9 9 8 , 70, 596-601.
(20) Bjartell, A.; Laine, S.; Pettersson, K.; Nilsson, E.; Lo¨ vgren, T.; Lilja, H.
Histochem. J. 1 9 9 9 , 31, 45-52.
(25) Takalo, H.; Mukkala, V.-M.; Mikola, H.; Liitti, P.; Hemmil¨a, I. Bioconjugate
Chem. 1 9 9 4 , 5, 278-82.
(26) Seveus, L.; Va¨isa¨la¨, M.; Syrja¨nen, S.; Sandberg, M.; Kuusisto, A.; Harju, R.;
Salo, J.; Hemmila¨, I.; Kojola, H.; Soini, E. Cytometry 1 9 9 2 , 13, 329-38.
(27) Mitrunen, K.; Pettersson, K.; Piironen, T.; Bjo¨ rk, T.; Lilja, H.; Lo¨ vgren, T.
Clin. Chem. 1 9 9 5 , 41, 1115-20.
(28) Merio¨ , L.; Petterssonm K.; Lo¨ vgren, T. Clin. Chem. 1 9 9 6 , 42, 1513-7.
(29) Takalo, H.; Hemmila¨, I.; Sutela, T.; Latva, M. Helv. Chim. Acta 1 9 9 6 , 79,
789-802.
(21) Scorilas, A.; Bjartell, A.; Lilja, H.; Moller, C.; Diamandis, E. P. Clin. Chem.
2 0 0 0 , 46, 1450-5.
(22) Saha, A. K.; Kross, K.; Kloszewski, E. D.; Upson, D. A.; Toner, J. L.; Snow,
R. A.; Black, C. D. V.; Desai, V. C. J. Am. Chem. Soc. 1 9 9 3 , 115, 11032-3.
(23) Prat, O.; Lopez, E.; Mathis, G. Anal. Biochem. 1 9 9 1 , 195, 283-9.
(24) Pierce, J. G.; Parsons, T. F. Annu. Rev. Biochem. 1 9 8 1 , 50, 465-95.
3194 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

Scheme 1. Synthesis of the Nonadentate Europium Chelate
Standard for Chorionic Gonadotropin (75/ 589), purchased from
the National Institute for Biological Standards and Control (United
Kingdom), in a pool of normal human male serum. All blood
samples used in this study were obtained from volunteers of the
staff, and the procedures followed were in compliance with the
Helsinki Declaration of 1975 as revised in 1996. For precision
studies, pooled heparin plasma samples were spiked with the hCG
standard material and frozen in aliquots at -70 °C until use. The
heparin whole blood samples were spiked similarly but analyzed
fresh.
Safety Considerations. All reagents of the organic syntheses
should be handled with caution. Thiophosgene and tin tetrachlo-
ride are toxic. Both of these reagents are toxic by inhalation, and
mechanical ventilation should be used. Tin tetrachloride is toxic
also in contact with skin and if swallowed; thiophosgene is harmful
in corresponding conditions. Personal protection should be used
when handling these reagents, and special care should be taken
when pouring the reaction mixture into the aqueous sodium
hydrogen carbonate because the reaction is vigorous. Appropriate
safety measures should also be followed when using the potassium
hydroxide and trifluoroacetic acids due to their corrosive nature.
Skin and eye contact and inhalation should be avoided with the
rest of the reagents as well.
Synthesis of {2 ,2 ′,2 ′′,2 ′′′-{[2 -(4 -Isothiocyanatophenyl)-
ethylim ino]bis(m ethylene)bis{4 -{[4 -(r-galactopyranoxy)-
phenyl]ethynyl}pyridine-6 ,2 -diyl}bis(m ethylenenitrilo)}-
tetrakis(acetato)}europium(III) (1 ). The novel nonadentate
label was prepared according to the steps shown in Scheme 1, as
described below. In flash chromatography (FC) we used Kieselgel
60 (0.040-0.063 or 0.063-0.200) silica gels (Merck).
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3195

(a) 4 -Iodophenyl 2 ,3 ,4 ,6 -Tetra-O-acetyl-r-galactopyrano-
side (3 ). 1,2,3,4,6-Penta-O-acetyl-â- -galactopyranose (12.0 g, 30.7
(f) {2,2′,2′′,2′′′-{[2-(4-Aminophenyl)ethylimino]bis(meth-
yle n e )b is {4 -{[4 -(r-ga la ctop yr a n oxy)p h e n yl]e th yn yl}-
pyridine-6 ,2 -diyl}bis(m ethylenenitrilo)}tetrakis(acetato)}-
europium(III) (9 ). A mixture of 8 (1.0 g, 0.544 mmol), 0.5 M
KOH in EtOH (55 mL), and H2O (25 mL) was stirred for 2 h at
room temperature. After evaporation, the residue was dissolved
in H2O (19 mL) and the pH adjusted to 6.5 with 6 M HCl. EuCl3
(237 mg, 0.647 mmol) in H2O (6.6 mL) was added within 15 min
and the pH maintained at 6.5 with solid NaCO3. After stirring the
reaction for 30 min, the pH was raised to 8.5 with 1 M NaOH and
the precipitate removed by centrifugation. The filtrate was then
treated with acetone and the precipitate collected by centrifuga-
tion. The product was washed with acetone and used in the next
step without further purification. The yield was 2.43 g.
D
mmol) and 4-iodophenol (9.5 g, 43.2 mmol) were dissolved in dry
ethanol-free chloroform (60 mL). Tin tetrachloride (10.8 mL, 92.5
mmol) was added, and the reaction was stirred at 62-64 °C for 7
h. Subsequently, the solution was poured into ice-cold aqueous
sodium hydrogen carbonate (600 mL) and diluted with dichlo-
romethane (600 mL). The solution was then filtered through Celite
(Aldrich), and the precipitate washed with additional 150 mL
of dichloromethane. The organic phase was rapidly washed with
ice-water (400 mL), dried (Na2SO4), and evaporated to give a
brown syrup. The product was purified by FC (Kieselgel
0.040-0.063) using AcOEt/ petroleum (1:3) to give 3 (yield 10.95
g, 65%).
(b) 4 -[(Trimethylsilyl)ethynyl]phenoxy 2 ,3 ,4 ,6 -Tetra-O-
acetyl-r-galactopyranoside (4 ). Compound 3 (10.9 g, 19.9
mmol), bis(triphenylphosphine)palladium(II) chloride (280 mg,
0.40 mmol), and CuI (152 mg, 0.80 mmol) were dissolved in a
mixture of dry Et3N (68 mL) and THF (78 mL), and the solu-
tion was deaerated with N2. (Trimethylsilyl)acetylene (3.5 mL,
24.8 mmol) was added, and the reaction stirred for 1.5 h at
room temperature. The mixture was then filtered and the fil-
trate evaporated. The residue was dissolved in CHCl3 (150 mL)
and washed with H2O (2 × 60 mL), dried (Na2SO4), and
evaporated. The product was purified by FC (Kieselgel 0.063-
0.200, AcOEt in petroleum ether, 10 f 20%). The yield was 7.43
g (66%).
(g) {2 ,2 ′,2 ′′,2 ′′′-{[2 -(4 -Isothiocyanatophenyl)ethylimino]-
bis(methylene)bis{4-{[4-(r-galactopyranoxy)phenyl]ethynyl}-
pyridine-6 ,2 -diyl}bis(m ethylenenitrilo)}tetrakis(acetato)}-
europium(III) (1 ). An aqueous solution of 9 (2.3 g, 1.75 mmol)
was added slowly (within 25 min) to a mixture of thiophosgene
(530 µL, 7.0 mmol), NaHCO3 (736 mg, 8.8 mmol), and CHCl3 (27
mL). After stirring for 1 h, the H2O phase was washed with CHCl3
(3 × 50 mL). The pH of the aqueous solution was then adjusted
to 7.0 with 1 M CH3COOH, and acetone was added. The product
was collected by centrifugation and washed with acetone to give
1 (2.26 g).
Labeling of Antibodies. The labeling of the detection antibody
was performed in 10 mmol/ L borate buffer, pH 8.6-9.0, using
50- and 150-fold molar excesses of the hepta- and nonadentate
chelates, respectively. The reactions were carried out overnight
at +4 °C for the heptadentate and at room temperature for the
nonadentate labels. Labeled antibodies were separated from
excess free chelate on Superdex 200 HR 10/ 30 or Superdex 200
HiLoad 26/ 60 gel filtration columns (Pharmacia Biotech) using
Tris-saline-azide (6.1 g/ L Tris, 9.0 g/ L NaCl, and 0.5 g/ L NaN3),
pH 7.75, as elution buffer. The fractions containing the antibody
were pooled and the europium concentrations measured against
a europium calibrator. The labeling degrees obtained were 3.6
and 3.5 Eu/ IgG for the hepta- and nonadentate labels, respectively.
Biotinylation of the capture antibody was performed in 50
mmol/ L NaHCO3, pH 9.8, using a 30-fold molar excess of the
biotinylation reagent (BITC) that was first dissolved in a small
volume of dimethylformamide. The reaction was carried out for
4 h at room temperature and the excess free reagent removed
using NAP-5 and NAP-10 (Amersham Pharmacia) or Superdex
200 HR 10/ 30 (Pharmacia Biotech) gel filtration columns, and the
Tris-saline-azide buffer, pH 7.75, for elution.
Finally, bovine serum albumin was added to a concentration
of 1 g/ L to the solutions containing the europium-labeled and the
biotinylated antibodies. The antibodies were stored at 4 °C.
Fluorescence P roperties of the Antibody Conjugates. The
fluorescence properties of the antibody-coupled europium chelates
were measured in the combined assay/ wash buffer used in the
hCG immunoassays, containing 5 mmol/ L HEPES, 2.1 g/ L NaCl,
0.1 mmol/ L EDTA, 0.055 g/ L Tween 20, and 1 g/ L Germall II,
pH 7.75. The excitation and emission spectra, fluorescence
intensities, and decay times were determined using a LS55
luminescence spectrometer (PerkinElmer Instruments), while the
molar extinction coefficients (absorbance) were determined with
(c) 4 -Ethynylphenoxy 2 ,3 ,4 ,6 -Tetra-O-acetyl-r-galacto-
pyranoside (5 ). Compound 4 (7.4 g, 14.2 mmol) was dissolved
in dry dichloromethane (110 mL) and deaerated with N2. Tet-
rabutylammonium fluoride (4.45 g, 17.02 mmol) was added to the
mixture and stirred for 30 min at room temperature, after which
the mixture was washed with 10% citric acid in H2O (70 mL) and
H2O (4 × 110 mL), dried (Na2SO4), and evaporated. The product
was purified by FC (Kieselgel 0.063-0.200, AcOEt in petroleum
ether, 20 f 40%) with a yield of 4.3 g (68%).
(d) Tetra(ter t-butyl) 2 ,2 ′,2 ′′,2 ′′′-{[2 -(4 -Aminophenyl)eth-
ylim ino]bis(m ethylene)bis{4 -{[4 -(2 ,3 ,4 ,6 -tetra-O-acetyl-r-
galactop yran oxy)p h en yl]eth yn yl}p yrid in e-6 ,2 -d iyl}bis -
(methylenenitrilo)}tetrakis(acetate) (7 ). Compound 6 was
synthesized as earlier described,29 dissolved (1.0 g, 1.0 mmol)
together with bis(triphenylphosphine)palladium(II) chloride (28
mg, 0.040 mmol) and CuI (15 mg, 0.078 mmol) in a mixture of
dry Et3N (10 mL) and THF (10 mL), and then deaerated with N2.
Compound 5 (1.08 g, 2.4 mmol) was added to the mixture, and
the reaction was stirred overnight at 53 °C, after which it was
filtered and the filtrate evaporated. The residue was dissolved in
CHCl3 (80 mL), washed with H2O (2 × 35 mL), dried (Na2SO4),
and then evaporated. The product was purified by FC (Kieselgel
0.063-0.200, MeOH in dichloromethane, first 0.5 f 2% and finally
10%). The yield was 1.34 g (77%).
(e) 2 ,2 ′,2 ′′,2 ′′′-{[2 -(4 -Aminophenyl)ethylimino]bis(meth-
ylene)bis{4 -{[4 -(2 ,3 ,4 ,6 -tetra-O-acetyl-r-galactopyranoxy)-
phenyl]ethynyl}pyridine-6 ,2 -diyl}bis(m ethylenenitrilo)}-
tetrakis(acetic acid) (8 ). A solution of 7 (1.3 g, 0.753 mmol) in
CF3COOH (13 mL) was stirred for 2 h at room temperature. After
evaporation without heating, the mixture was triturated with Et2O
(50 mL) and filtered. The yield was 1.3 g.
3196 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

a UV-2100 spectrophotometer (Shimadzu). Additionally, decay
times were also measured from antibody conjugates bound to solid
phase, either covered with 30 µL of the combined assay/ wash
buffer or after aspirating and drying of the wells. The solid-phase
determinations were performed using a Cary Eclipse fluorescence
spectrophotometer (Varian).
Table 1. Fluorescence Properties of the
Antibody-Coupled Chelates
τsolid phase (µs)
λexc
λem
τliquid phase
(µs)
(nm) (nm)
weta
dry
ꢀΦ
Φ
9-dentate
7-dentate
325
329
615
615
1000
390
1000
490
1150 4787 0.127
990 1337 0.042
P reparation of the All-in-One Dry Reagent Wells. The
streptavidin coating of the single wells was performed through
physical adsorption. A 750-ng sample of streptavidin was added
per well in 150 µL of coating buffer, containing 100 mmol/ L NaH2-
PO4 and 50 mmol/ L citric acid, pH 5.0, and incubated overnight
at room temperature. The coated wells were washed twice (5
mmol/ L Tris, 154 mmol/ L NaCl, 5 g/ L Tween 20, and 1 g/ L
Germall II, pH 7.75) and saturated overnight at room temperature
with 300 µL/ well of a solution containing 26.6 g/ L bovine serum
albumin, 50 mmol/ L Tris, 154 mmol/ L NaCl, 330 mmol/ L
a
30 µL of the combined assay/ wash buffer per well.
time, 1 s (i.e., counts resulting from 1000 sequential excitations
were integrated for each measurement).
To compare the sensitivity of solid-phase and dissociation-
enhanced measurements of the novel nonadentate label, some of
the assay cups first measured dry in the Aio immunoanalyzer were
collected and remeasured using the commercial dissociation-
enhanced lanthanide fluoroimmunoassay (DELFIA) principle
(PerkinElmer Life Sciences/ Wallac Oy).30 In DELFIA, the eu-
ropium ions are dissociated from the original, nonfluorescent
chelates at low pH using a special enhancement solution. The
enhancement solution also contains â-diketone, detergent, and
chelator that interact to form a strongly fluorescent, micellar
complex with the dissociated lanthanide ions. For this purpose,
200 µL of the enhancement solution (DELFIA reagent) was
dispensed in each well and incubated for 60 min at room
temperature with slow shaking (>90% dissociation obtained for
the nonadentate chelate). The fluorescence counts were measured
using a Victor 1420 multilabel counter (PerkinElmer Life Sci-
ences/ Wallac Oy) with the default settings for europium measure-
ment.
D
-sorbitol, and 0.5 g/ L NaN3, pH 7.0. After saturation, the wells
were aspirated and dried for 3 h in a dry-condition cabinet (35
°C, relative air humidity 5%).
For preparing the assay-specific dry reagent wells, 400 ng
of biotinylated capture antibody was added in the wells in 50 µL
of coating buffer containing 50 mmol/ L Tris, 154 mmol/ L
NaCl, 20 µmol/ L DTPA, 0.1 g/ L Tween 40, 5 g/ L BSA, 0.5 g/ L
bovine γ-globulin, 0.5 g/ L NaN3, and 0.02 g/ L cherry red,
pH 7.75, and incubated overnight at room temperature. The
wells were again washed and the antibodies covered with a
protective solution, 40 µL/ well, containing 37.5 mmol/ L Tris,
120 mmol/ L NaCl, 0.375 g/ L NaN3, 0.6 g/ L bovine γ-globulin, 25
g/ L bovine serum albumin, 50 g/ L D-trehalose, 0.1 g/ L native
mouse IgG, 0.05 g/ L denatured mouse IgG, 2 g/ L casein, and
10 g/ L PVA 6000, pH 7.75. Drying of the wells was performed
overnight in a dry-condition cabinet at 35 °C with 5% relative air
humidity. The labeled detection antibody (200 ng/ well) was
applied on top of the protective layer in a 1-µL drop of a buffer
containing 50 mmol/ L Tris, 154 mmol/ L NaCl, 0.5 g/ L NaN3,
RESULTS
Syntheses. The novel nonadentate europium(III) chelate 1
was prepared according to the route shown in Scheme 1. The
organometallic coupling of aryl bromide 6 29 with the terminal
acetylene (4-ethynylphenoxy)-2,3,4,6-tetra-O-acetyl-R-galactopyra-
noside (5 ) gave compound 7 . Compound 5 was prepared starting
from 4-iodophenol and 1,2,3,4,6-penta-O-acetyl-â-
The reaction of 1,2,3,4,6-penta-O-acetyl-â- -galactopyranose and
125 g/ L D-trehalose, and 5 g/ L bovine serum albumin, pH 7.75,
and immediately dried with a steam of warm air. The dry
reagent wells were stored protected from humidity at room
temperature.
D-galactopyranose.
D
Immunoassay for hCG. The noncompetitive, one-step im-
munoassays for hCG were performed on a fully automated Aio
immunoanalyzer (Innotrac Diagnostics) that allows time-resolved
fluorescence measured directly from a solid phase. In the
automated assay procedure, 10 µL of sample (whole blood or
plasma) and 20 µL of the combined assay/ wash buffer were
dispensed in the dry reagent well and incubated for 15 min (i.e.,
until equilibrium) with slow shaking at 36 °C. The wells were
washed and aspirated a total of 6 times, with the last aspiration
adjusted so that the wells were either aspirated dry or a well-
defined amount of the assay/ wash buffer (from 10 ( 1 to 150 (
10 µL) was left in the well. For dry measurements, a steam of hot
(95 °C) air was blown to the assay cups for 40 s prior to
measurement to ensure dryness of the wells, while in the wet
measurement mode, this step was omitted. The measurement of
europium fluorescence from the well surface was carried out using
the default measurement settings of the immunoanalyzer: excita-
tion wavelength, 340 nm; emission wavelength, 615 nm; delay time,
400 µs; window time, 400 µs, cycling time, 1 ms; measurement
4-iodophenol with tin tetrachloride in CHCl3 gave a good yield of
R-anomer 4-iodophenyl 2,3,4,6-tetra-O-acetyl-R-galactopyranoside
(3 ).31 The 4-iodo atom of 3 reacted with (trimethylsilyl)acetylene
in the presence of a palladium catalyst and copper(I) iodide. The
silyl protection was easily removed by tetrabutylammonium
fluoride to give 5 . The ester groups of 7 were hydrolyzed with
trifluoroacetic acid. The acetyl groups of 8 were saponified, and
the europium(III) chelate was prepared by stirring the tetrakis-
(acetic acid) with EuCl3 in slightly acidic conditions. Finally, the
amino group of 9 was activated with thiophosgene.
Fluorescence P roperties of the Antibody Conjugates. The
excitation (λexc) and emission (λem) maximums, fluorescence yields
(ꢀΦ), and quantum yields (Φ) of the nona- and heptadentate
europium chelates were determined using the combined assay/
wash buffer for dilution (Table 1). Both chelates had an excitation
(30) Hemmila¨, I.; Dakubu, S.; Mukkala, V.-M.; Siitari, H.; Lo¨ vgren, T. Anal.
Biochem. 1 9 8 4 , 137, 335-43.
(31) Blanc-Muesser, M.; Driquez, H. J. Chem. Soc., Perkin Trans. 1 1 9 8 8 , 3345-
51.
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3197

maximum in the range of 320-340 nm and a sharp emission at
615 nm, facilitating the use of the default settings for europium
measurement in the Aio immunoanalyzer and Victor 1420 multi-
label counter. The fluorescence and quantum yields were,
however, more than 3-fold higher for the nonadentate chelate
compared to the heptadentate one.
The fluorescence decay times (τ) of the label-antibody
conjugates were measured both free in liquid phase and bound
to the surface of an assay well, the latter either wet (30 µL of
buffer/ well) or after aspirating and drying of the wells (Table 1).
Unlike the heptadentate label, the fluorescence lifetime of which
significantly increased after drying, the nonadentate europium
chelate demonstrated rather uniform and long standing (in the
range of ∼1 ms) decay times in all the different measure-
ment conditions, thus indicating good tolerance against aqueous
quenching.
Measurement of the decay times also facilitated estimation of
the number of water molecules, q, in the first coordination sphere
of the europium ions using the equation derived by Horrocks and
Sudnick32 and later specified by Beeby et al.:33
q ) A(τ-1
- τ-1
- 0.25)
D2O
H2O
Figure 2. Moisture tolerance of the nonadentate (A) and hepta-
dentate (B) chelates in hCG assay. The one-step assays were
performed using 25 (black box) or 1000 (gray box) IU/L hCG.
Fluorescence was measured either directly from aspirated (moist) well
surface, from wells containing 10-150 µL of the combined wash/
assay buffer, or from aspirated wells dried with hot air. The relative
fluorescence signals are indicated with columns and the relative
signal-to-noise ratios with lines. The calculations are based on four
replicate measurements.
where A is a proportionality constant suggested to be 1.2 waters‚
ms for europium33 and τ is the experimental fluorescence lifetime
in milliseconds. Although the exact value for q can be calculated
only based on parallel measurements in both ordinary and
deuterated water, good estimates can be obtained using an average
decay constant of 0.5 ms-1 (corresponding to a decay time of 2
ms) for a europium(III) chelate in D2O.34,35 By this adaptation, q
computes to be ∼0.3 for the antibody-coupled nonadentate chelate
and ∼2.2 for the corresponding heptadentate one, both measured
free in the aqueous solution. In the case of the heptadentate label,
however, the binding of the label-antibody conjugate onto the well
surface resulted in a slight increase in the label’s decay time, thus
decreasing the calculated q to ∼1.5. Such a change may imply,
for example, some kind of a structural change in the label-
antibody conjugate that leads to the exclusion of some of the
coordinated water molecules or, alternatively, protection of the
chelate by the proximity of the surface.
cence level could be recovered from the wells containing liquid
(Figure 2). For the nonadentate chelate, however, no decrease in
fluorescence was observed when the wells were measured after
aspiration, and ∼60% of maximal signal level could be obtained
also from wells containing buffer. Furthermore, the intensity of
fluorescence and the corresponding signal/ noise ratios were
independent of the amount of buffer per well for both chelates in
the wet measurement mode. Therefore, we selected a residual
buffer volume of 30 µL for subsequent experiments.
Calibration Curves. The calibration curves and analytical
detection limits (calculated as the mean of background + 3 SD)
obtained with the dry, moist (aspirated wells), and wet (30 µL of
buffer/ well) measurements using either the nona- or heptadentate
europium chelates as labels are shown in Figure 3. Consistent
with the fluorescence intensity measurements, the analytical
detection limits obtained in the different moisture conditions were
significantly divergent for the assays using the heptadentate label
(1.7, 2.4, and 5.1 IU/ L hCG for the dry, moist, and wet measure-
ments, respectively) compared with the rather consistent detection
limits calculated for the ones using the nonadentate label (0.7,
0.8, and 0.9 IU/ L hCG, in the same order as above). The
differences in the detection limits were, however, generally smaller
than could have been predicted from the fluorescence intensity
studies, mainly because the background fluorescence of the assays
followed the change in the specific signal.
Moisture Tolerance of the Europium Chelates in hCG
Immunoassay. To assess the moisture tolerance of the two
chelates in a noncompetitive assay for hCG, we performed the
immunoassays as usual and then measured the fluorescence
intensities from the assay wells in different moisture conditions.
Accordingly, fluorescence was measured directly from a set of
aspirated but still moist wells, from wells containing different
volumes (10-150 µL) of the combined assay/ wash buffer, and
from aspirated wells that were further dried with hot air.
As expected, the signal intensity of the heptadentate chelate
was already decreased by 50% when measured from an aspirated
instead of a dry surface, and only ∼20% of the maximal fluores-
(32) Horrocks, W. D.; Sudnick, D. R. J. Am. Chem. Soc. 1 9 7 9 , 101, 334-40.
(33) Beeby, A.; Clarkson, I. M.; Dickins, R. S.; Faulkner, S.; Parker, D.; Royle,
L.; de Sousa, A. S.; Williams, J. A.; Woods, M. J. Chem. Soc., Perkin Trans.
2 1 9 9 9 , 493-504.
(34) Horrocks, W. D.; Sudnick, D. R. Acc. Chem. Res. 1 9 8 1 , 14, 384-92.
(35) Byden, C. C.; Reilley, C. N. Anal. Chem. 1 9 8 2 , 54, 610-15.
For comparison, a calibration curve was run for the assay
employing the nonadentate label also using the DELFIA principle
3198 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

Table 2. Assay Variability
heparin whole blood
heparin plasma
a
a
b
within-run CV%
within-run CV%
between-run CV%
25/ 500/ 5000 IU/ L hCG
25/ 500/ 5000 IU/ L hCG
25/ 500/ 5000 IU/ L hCG
9-dentate
7-dentate
dry
8.0/ 5.3/ 3.5
4.7/ 6.2/ 4.0
8.1/ 3.7/ 2.1
7.0/ 8.3/ 8.3
15/ 6.9/ 3.8
4.6/ 4.0/ 1.5
3.5/ 4.7/ 2.8
8.9/ 2.7/ 2.4
7.2/ 4.8/ 2.4
14/ 4.9/ 2.4
4.0/ 4.2/ 4.7
7.9/ 4.9/ 4.3
wet
DELFIAc
dry
wet
a
n ) 12. b Duplicate samples run for 12 days; 2 runs per day. c Four wells from the total of 72 were excluded from calculations due to falsely
increased signal intensities.
Furthermore, although very good results were obtained by
measuring the hCG assay wells immediately after washing and
aspiration (without drying), this measurement mode was, in our
opinion, potentially too susceptible to environmental influences
and disturbances resulting, for example, from variation in air
humidity, temperature, or aspiration efficiency. Since these kinds
of changes can affect the moisture level in the cups and, therefore,
compromise the accuracy of the fluorescence detection, we
decided to omit this mode of measurement in the subsequent
experiments and concentrate on the more robust wet and dry
measurements.
Assay P recision and Recovery. The variability and recovery
of the hCG assay were studied by adding known amounts of
purified hCG to hCG-free human heparinized whole blood and
plasma samples (Table 2). The highest within-assay variation, 14-
15% at the hCG concentration of 25 IU/ L, was seen for the wet
measurements of the heptadentate label, being a consequence of
the decreased signal level in these measurements due to aqueous
quenching. For the nonadentate label, the within-assay CVs were
in the range of 1.5-8.0% for the wet and dry measurements and
2.1-8.9% for the DELFIA measurements, although in the latter
case 4 wells (so-called “flyers”) from a total of 72 were excluded
from the calculations due to significantly (falsely) increased signal
intensities. The between-assay variability was tested for the
nonadentate chelate only, and it was found to be <8% for both
wet and dry measurements.
The analytical recovery of the rapid hCG immunoassay is
shown in Table 3. Recoveries were in the same range for whole
blood and plasma, being 92-114 and 86-122% for the assays using
the nona- and heptadentate labels, respectively, indicating the
accuracy of the method and an absence of interference from whole
blood. In all, no differences were detected between whole blood,
plasma, and serum samples in any of the measurements. The
average background fluorescence counts using any of these
matrixes remained below 700 using the heptadentate label and
below 600 using the nonadentate label, of which ∼150 fluores-
cence counts originated from the polystyrene cups and the
instrument itself.
Figure 3. Calibration curves (solid symbols) and precision profiles
(open symbols) using the nonadentate (A) and heptadentate (B)
chelates as labels in the hCG assay. Fluorescence was measured
either from aspirated (moist) wells, from wells containing 30 µL of
the combined wash/assay buffer, or from aspirated wells dried with
hot air. The values shown are the mean of four replicates. The
analytical sensitivities of the assays were calculated as the mean of
background (12 replicates) + 3 SD and are indicated with dotted lines.
for detection (Figure 3A). In the current assay format, however,
the use of this measurement principle led to an increase in the
background level as well as an increased well-to-well variation,
and the analytical detection limit of 1.4 IU/ L hCG obtained using
the DELFIA method did not exceed those of the direct surface
measurements.
DISCUSSION
New labels and assay techniques are needed to keep up with
the expectations set for the analytical performance of future POCT.
Time-resolved fluorometry, which has for long been applied in
both clinical routine and research applications (for review, see
Analytical Chemistry, Vol. 75, No. 13, July 1, 2003 3199

Table 3. Analytical Recovery of HCG (n ) 12)
heparin plasma
heparin whole blood
original hCG
(IU/ L)
added hCG
(IU/ L)
measured
(IU/ L)
recovery
(%)
measured
(IU/ L)
recovery
(%)
9-dentate
7-dentate
dry
wet
dry
wet
0
0
0
0
25/ 500/ 5000
25/ 500/ 5000
25/ 500/ 5000
25/ 500/ 5000
27/ 566/ 5549
24/ 546/ 5485
27/ 503/ 4464
24/ 463/ 4733
109/ 113/ 111
97/ 109/ 110
107/ 101/ 89
97/ 93/ 95
27/ 572/ 5249
23/ 534/ 5024
31/ 538/ 4321
23/ 469/ 4705
107/ 114/ 105
92/ 107/ 100
122/ 108/ 86
93/ 94/ 94
refs 36 and 37), provides an excellent way of creating highly
sensitive yet robust assay formats suitable for quantitative and
rapid testing. The measurement ranges and sensitivities obtainable
with the time-resolved fluorescence technology are generally
orders of magnitude higher than for conventional fluorescence,
deriving from the unique long-lived emission properties of the
lanthanide ions and the effective elimination of the rapidly
decaying background fluorescence originating from serum pro-
teins, plastic ware, and light scattering.15-18
the dry mode (from 1.7 to 5.1 IU/ L hCG), while the corresponding
loss was <30% for the nonadentate label with the analytical
detection limit remaining below 1 IU/ L hCG.
The nonadentate chelate was also measured using the com-
mercial DELFIA measurement principle, in which a separate step
for fluorescence enhancement is employed.30 However, this
method of detection was not fully compatible with the current
assay format and resulted in an increased background level and
assay variation. Mostly this was due to the gentle washing
procedure that was optimized for the direct surface detection at
the bottom of the well. In the DELFIA measurements, however,
the fraction of label that was nonspecifically bound to the sidewalls
of the wells could be dissociated and detected, occasionally also
resulting in significant aberrations (so-called “flyer” wells). How-
ever, the reproducibility and sensitivity of the DELFIA measure-
ments could most likely have been improved using more efficient
washings, although they were not applied in this study.
Although increasing the number of chromophores generally
tends to increase the hydrophobicity of the chelate and, therefore,
the extent of nonspecific binding, no such increase in background
was observed for the novel chelate when compared with the
heptadentate chelate in direct surface measurements. Most likely
this was due to the balancing effect of the two hydrophilic
R-galactose side groups, which also rendered the chelate soluble
in aqueous solvents. Most importantly, the binding activity of the
detection antibody was preserved after conjugation with the new
label, even though it must be noted that since the ultimate
sensitivity of the hCG assay was not a subject of investigation for
this study, the labeling degree of the detection antibody was kept
relatively low (3.6 and 3.5 europiums/ IgG molecule using the
hepta- and nonadentate chelates, respectively). Owing to the gentle
coupling via the chelate’s isothiocyanato groups and the antibody’s
primary amino groups, however, it is possible that the labeling
degree of the antibody could be multiplied without a significant
change in its binding characteristics, as shown by earlier studies
using corresponding low molecular weight lanthanide chelates.18,30
Even so, the assay sensitivities readily obtained using the
nonadentate label were more than sufficient for the intended use
for detection of pregnancy, with the within- and between-assay
CVs below 8% at the common hCG cutoff concentration of 25 IU/
L38 in both wet and dry measurements. Furthermore, the assays
were linear at least up to 5000 IU/ L hCG, thus covering almost 4
orders of magnitude.
In the current study, we focused on time-resolved surface
measurement of two intrinsically fluorescent europium chelates,
a previously produced heptadentate and a novel nonadentate, and
the applicability of them in a single-well-based POC assay platform.
Although successfully used in several different bioaffinity
applications,9,10,14,20,26-28 the heptadentate label was, however,
known to be sensitive to aqueous quenching due to the molecular
structure that allows water molecules in the first coordination
sphere of the lanthanide ion. In the nonadentate label, however,
all the coordination sites of the europium ion are occupied by
carboxylate and amine moieties, providing a very high thermo-
dynamic and kinetic stability as well as minimal nonradiative
energy transfer to the quencher molecules. The measured
fluorescence decay times of the two chelates were consistent with
these structural details. While significant quenching was observed
for the antibody-conjugated heptadentate label in the presence of
water molecules, the fluorescence lifetime of the nonadentate label
was only slightly decreased and remained in the range of ∼1 ms.
Correspondingly, a significantly higher quantum yield (Φ), i.e.,
the portion of excited label molecules that emit light, was
measured for the antibody-conjugated nonadentate label (12.7%)
free in an aqueous solvent compared to the heptadentate one
(4.2%).
Furthermore, two separate chromophores were incorporated
in the nonadentate label to facilitate more efficient absorption of
excitation light. Combined with the effective shielding from water
molecules, almost a 4 times higher total fluorescence yield was
measured for the antibody-conjugated nonadentate label compared
to the heptadentate one. As expected, the difference in the
fluorescence yields was directly reflected in the signal-to-noise
ratios obtained in the wet measurements and, therefore, in the
detection limits of the hCG assay, although the degree of
background discrimination was slightly enhanced by the concur-
rent decrease of background fluorescence as well. Taken together,
a 3-fold deterioration of the analytical detection limit was observed
for the heptadentate label when measured in the wet instead of
Even though a number of intrinsically fluorescent lanthanide
chelates have been developed and applied in different bioaffinity
applications during the past years, the current nonadentate chelate
(36) Soini, E.; Hemmila¨, I.; Dahlen, P. Ann. Biol. Clin. (Paris) 1 9 9 0 , 48, 567-
71.
(37) Dickson, E. F.; Pollak, A.; Diamandis, E. P. Pharmacol. Ther. 1 9 9 5 , 66,
207-35.
(38) Alfthan, H.; Bjo¨ rses, U.-M.; Tiitinen, A.; Stenman, U.-H. Scand. J. Clin.
Lab. Invest. Suppl. 1 9 9 3 , 216, 105-13.
3200 Analytical Chemistry, Vol. 75, No. 13, July 1, 2003

is one of the few that can be measured wet without significantly
compromising the fluorescence yield and that fulfills all the strict
requirements set for use in bioaffinity applications. However, in
addition to increasing the number of lanthanide-coordinating
groups in the ligand structure, other alternatives exist to similarly
avoid aqueous quenching of the fluorescence. One of the com-
monest approaches is to incorporate the fluorescent chelates into
polymeric latex or polystyrene particles and thus physically protect
them from environmental effects. A major advantage of this
approach is that large numbers of label molecules can be included
in the particles, thus also offering a way of improving assay
sensitivity by signal amplification.39,40 Correspondingly, several
other types of novel particulate labels have been applied in
ultrasensitive bioassays, including quantum dots,41,42 down-43 and
upconverting44 phosphors, and plasmon-resonant particles.45 In
general, however, the particulate labels suffer from several
limitations that also have obstructed their use in routine applica-
tions. Due to their relatively large size (inner diameters usually
100 nm or higher), the particles diffuse very slowly and have an
increased tendency of aggregation and nonspecific binding,
resulting in slow kinetics and assay variation. In some cases, the
particles may also cause sterical hindrance to the binding of
biomolecules. The assay procedures using these labels are usually
complicated and require, in addition to the prolonged incubation
times, separate steps for incubating the antigen and label with
the solid-phase antibody.
Due to the reasons described above, heterogeneous assays
utilizing the particulate labels are not easily automated and are
therefore presently mostly used for research purposes. However,
the high-sensitivity particulate labels do have potential use in
simple immunochromatographic assay formats where the diffusion
distances are short and the assay kinetics little affected by the
slow diffusion properties of the particles. For example, prelim-
inary results describing the use of upconverting phosphors in
lateral flow assay systems have recently been reported,46,47 with
an analytical detection limit (background + 2 SD) of 10 pg/ mL
hCG in a Hepes-based buffer, equal to ∼0.9 IU/ L hCG.46 However,
due to the limitations inherent to the labels and the assay format,
the reproducibility and linearity of this and most of the current
immunochromatographic assays do not yet correspond to those
of the more advanced immunoassay formats.
techniques, analytical information can, however, be rapidly ob-
tained in POC situations also without sacrificing the sensitivity
and quantitativeness of the immunoassays, as shown in this study.
By using the novel intrinsically fluorescent nonadentate europium
chelate and the all-in-one dry reagent chemistry, we were able to
diminish the number of assay steps to three, comprising the one-
step incubation, washing, and direct measurement of time-resolved
fluorescence from a wet well surface. Combined with the inde-
pendency of the signal level both on the sample type (i.e.,
unprocessed whole blood versus plasma and serum) and on the
amount of residual buffer per well (10-150 µL), the current assay
format provides an extremely robust and rapid, yet sensitive and
fully quantitative immunoassay procedure that can easily be
automated. Although the assays were now run with the midsized
Aio immunoanalyzer, a simpler and smaller instrument, targeted
for use in rapid diagnostics for example at the physician’s office,
is currently under development. Further improvements of the
assay platform will also include, in addition to applying it for other
analytes, a further reduction in the total assay time. Although we
used an incubation time of 15 min in the current study, the small
reaction volumes, effective shaking, increased reaction tempera-
ture (36 °C), carefully selected antibodies, and high solid-phase
binding capacity facilitated more than 80% of equilibrium to be
reached in only 5 min. Therefore, the use of a 5-min incubation
time in the current assay format should not significantly affect
the assay performance while, however, remarkably shortening the
time to results.
In conclusion, the newly synthesized 9-dentate label provided
several advantages for the development of a robust and sensitive
POC immunoassay format with direct surface measurement of
time-resolved fluorescence. The novel label also has a high
potential for adaptation to many different applications requiring
spatial resolution, including immunohistochemistry, in situ hy-
bridization, and microarray analysis.
ACKNOWLEDGMENT
This work was supported by the National Technology Agency
of Finland (TEKES) and by grants from the Paulo Foundation,
the Instrumentarium Science Foundation, the Alfred Kordelin
Foundation, and the Finnish Cultural Foundation. We gratefully
acknowledge Annika Salminen and Pirjo Laaksonen for excellent
technical assistance, Piitu Jauria and Tero Soukka for helpful
discussions, Harri Hakala for performing the liquid-phase mea-
surements of the chelate fluorescence properties, Jarmo Rainaho
for help in preparing the all-in-one assay wells, and Tom Javen
for making the required modifications in the immunoanalyzer
programming. Presented in part at the AACC Annual Meeting,
Orlando, FL, 2002, and the 18th ICCC, Kyoto 2002.
Sufficient analytical quality of results is, however, extremely
important regardless of whether the assays are performed in
central laboratories or in POC conditions, although this is often
overlooked in the latter case. With novel labels and assay
(39) Hall, M.; Kazakova, I.; Yao, Y. M. Anal. Biochem. 1 9 9 9 , 272, 165-70.
(40) Soukka, T.; Paukkunen, J.; Ha¨rma¨, H.; Lo¨ nnberg, S.; Lindroos, H.; Lo¨ vgren,
T. Clin. Chem. 2 0 0 1 , 47, 1269-78.
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SUPPORTING INFORMATION AVAILABLE
1 9 9 8 , 281, 2013-6.
(43) Beverloo, H. B.; van Schadewijk, A.; Zijlmans, H. J.; Tanke, H. J. Anal.
Biochem. 1 9 9 2 , 203, 326-34.
The NMR and mass spectra of compounds 3 -8 , and the UV
and mass spectra of compounds 9 and 1. This material is available
free of charge via the Internet at http:/ / pubs.acs.org.
(44) Zijlmans, H. J.; Bonnet, J.; Burton, J.; Kardos, K.; Vail, T.; Niedbala, R. S.;
Tanke, H. J. Anal. Biochem. 1 9 9 9 , 267, 30-6.
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(46) Hampl, J.; Hall, M.; Mufti, N. A.; Yao, Y. M.; MacQueen, D. B.; Wright, W.
Received for review January 3, 2003. Accepted April 9,
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AC0340051
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