Luminescent probes for ultrasensitive detection of nucleic acids

Article abstract of DOI:10.1021/bc900403n

Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescencebased molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanidebased hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications.

Full text of DOI:10.1021/bc900403n

Bioconjugate Chem. 2010, 21, 319–327
319
Luminescent Probes for Ultrasensitive Detection of Nucleic Acids
Lev N. Krasnoperov,† Salvatore A. E. Marras,*,‡ Maxim Kozlov,‡ Laura Wirpsza,†,‡ and Arkady Mustaev‡
Department of Chemistry and Environmental Sciences, New Jersey Institute of Technology, 151 Tiernan Hall, University
Heights, Newark, New Jersey 07102, and PHRI Center, New Jersey Medical School, Department of Microbiology and
Molecular Genetics, University of Medicine and Dentistry of New Jersey, 225 Warren Street, Newark, New Jersey 07103.
Received September 14, 2009; Revised Manuscript Received November 11, 2009
Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention
were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved
acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance
of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1
pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-
based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-
based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all
type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers
and small compounds that are used in a variety of biological applications.
Two commonly used classes of lanthanide chelates are
diethylenetriaminepentaacetic acid (DTPA) and tetraethylene-
tetraminohexaacetic acid (TTHA). These chelates attached to
7-animo quinolones (10-17) are known as DTPA and/or
TTHA-cs124 derivatives. The advantage of these classes of
compounds is their high quantum yield, high solubility in water,
and the possibility of introducing chemical modifications in the
fluorophore to spectrally optimize the transfer of energy to the
lanthanide and to enable the attachment of a cross-linking group.
A number of methods for the conjugation of these chelates to
biomolecules have been suggested. One of them is to use the
dianhydride form of DTPA, in which one of the anhydrides
modifies the amino group of the chromophore, while the other
anhydride reacts with amino group of the biomolecule (17).
Even though this approach is technically simple, it raises
concerns about the side reactions (modification of other nu-
cleophilic groups) due to the high reactivity of anhydrides. The
second approach takes advantage of the conjugation of one of
the DTPA anhydride groups with the cs124 moiety, followed
by reaction of the remaining anhydride with the diamine. The
unmodified amino group of the resulted adduct can then be
converted to an amino-reactive isothiocyano or thiol-reactive
groups (12). This mode of attachment of the cross-linking group
weakens the retention of the lanthanide within the chelate by
eliminating one ligating carboxylate, and it also reduces the
brightness of the lanthanide (14) (30% to 1000%) due to the
quenching effect of the additional coordinated water molecule.
These factors restrict in ViVo applications where high concentra-
tion of metal scavengers is an issue (e.g., intracellular imaging).
Analogous derivatives of the fluorophore coumarine have been
suggested and used in biophysical studies (18). However,
compared to their quinolone counterparts, they are less bright
and do not support terbium (Tb) luminescence.
INTRODUCTION
Some lanthanide chelates possess the unique spectral property
known as luminescence (1-9). The principle features of
luminescence are long-lived excited states (in the microsecond
to millisecond range) and narrow emission spectra. Temporal
and spectral gating enables unusually sensitive detection of
lanthanide emission, even in samples that exhibit significant,
short-lived autofluorescence (e.g., biological specimens or
tissues). These compounds are therefore potentially useful in a
wide variety of technical and biological tasks, such as tracing
analysis, immunoanalysis, tissue-specific imaging, and detection
of single molecules in living cells. Despite the benefits offered
by this technique, its widespread use is impeded by the limited
choice of instrumentation and the extremely high price of
commercial probes. Yet, existing lanthanide-based probes could
be improved to meet the requirements of new challenging
applications.
Due to their very low absorbance (ε < 1 M-1 cm-1),
lanthanide ions have to be “pumped”, which is achieved by
tethering the lanthanide ion (through chelation) to an appropriate
organic fluorophore (antenna). In such constructs, the energy
absorbed by the antenna is transferred radiationlessly to the
lanthanide, which finally emits the light. The chelating group
also shields the lanthanide ion from the quenching effects of
the medium, increasing the quantum yield of the probe.
Development of new luminescent probes is challenging, since
the transfer of energy from the antenna to the lanthanide is
complex (a process not yet well understood) and very sensitive
to subtle structural variations in the fluorophore. Another
challenge is the necessity of combining three functional units
within the same reporting probe: an antenna, a chelated
lanthanide, and a cross-linking group (for attachment to the
biomolecule of interest). This requires a complex synthetic
strategy, eventually leading to compounds whose size often
exceeds 1000 Da.
To further perfect lanthanide probes, we now report the
development of two novel approaches for the introduction of
cross-linking groups into DTPA and/or TTHA-cs124 chelates
by modification of the chromophore moiety. We demonstrate
that the synthesized luminescent probes (Chart 1) are more
resistant to EDTA challenge than previously described DTPA-
cs124-based probes, and we demonstrate that these new probes
are highly luminescent. These compounds were validated as
* To whom correspondence should be addressed: marrassa@
umdnj.edu.
† New Jersey Institute of Technology.
‡ University of Medicine and Dentistry of New Jersey.
10.1021/bc900403n  2010 American Chemical Society
Published on Web 01/19/2010

320 Bioconjugate Chem., Vol. 21, No. 2, 2010
Krasnoperov et al.
Chart 1. Structure of Reactive Luminescent Probes and Reference Compounds Used in This Study
luminescent labels by including them in molecular beacon
probes, which are widely used for DNA and RNA detection
(19). The resulting sensitivity of these molecular beacons was
between 0.5 and 1 pM, which is the most sensitive level of
detection ever reported for nonamplified DNA detection systems.
(7 mmol) of 1,3-phenylenediamine was added, followed by
incubation at 110 °C for 6 h. Under these conditions, four
fluorescent products were detected by thin layer chromatography
in ethyl acetate as developing solvent: Rf ) 0.90 (green-blue
fluorescence); Rf ) 0.45 (blue fluorescence); Rf ) 0.30 (blue
fluorescence); and Rf ) 0.03 (green-blue fluorescence). This
mixture was diluted with 30 mL of 0.05 M aqueous NaOH and
extracted with ether (2 × 40 mL). The aqueous layer was
separated and treated as described below (section 2). The organic
layer was extracted with 1 vol 0.1 M citric acid, and then
collected, dried over anhydrous sodium sulfate, and evaporated
in vacuo. The residue was subjected to silica gel chromatography
on a 40 mL column, using a hexane/acetone mixture (3:1) as
eluent. Fractions corresponding to the products migrating with
Rf ) 0.45 (product II) were collected and evaporated to dryness.
The residue was then washed with chloroform and dried. Yield
EXPERIMENTAL PROCEDURES
Synthesis. The following reagents were purchased from
Aldrich: diethylenetriaminepentaacetic acid dianhydride (DTPA),
triethylamine, 1,3-phenylenediamine, ethyl 4,4,4-trifluoroac-
etoacetate, ethylacetoacetate, 1,3-dicyclohexylcarbodiimide (DCC),
ethylenedianime, N-trityl-1,6-diaminohexane, methylbromac-
etate, anhydrous dimethylformamide and dimethylsulfoxide,
1-butanol, ethylacetate, chloroform, acetonitrile, ethanol, sodium
and potassium hydroxide, ZnCl2, Na2SO4, Na2CO3, acetic acid,
citric acid, thiocarbonyldiimadazole, TbCl3, EuCl3, SmCl3 and
DyCl3, silica gel TLC plates on aluminum foil (200 µm layer
thick with a fluorescent indicator). Only distilled and deionized
water (18 MΩ cm-1) was used. All experiments, including the
preparation and use of lanthanide complexes, were performed
either in glassware washed with mixed acid solution and rinsed
with metal-free water or in metal-free plasticware purchased
from Bio-Rad. All chemicals were the purest grade available.
Probe 1 (Scheme 1A). 1. 7-Amino-4-trifluoromethyl-3-
carbomethoxymethyl-2(1H) quinolone (II) (cs124CF3-CH2-
COOCH3). 4,4,4-Trifluoroacetoacetate (2.2 mL, 15 mmol) and
KOH (0.86 g, 15 mmol) were mixed in 7 mL of dimethylfor-
mamide and stirred at 40 °C until dissolved. To this mixture,
1.5 mL of methylbromoacetate was added and the solution was
incubated overnight at room temperature. Three hundred mil-
ligrams (5.2 mmol) of KOH was then added, and incubation
continued at 60 °C for 1 h. This mixture was diluted by the
addition of 20 mL of water and was then extracted with
chloroform. The organic layer was collected, dried over
anhydrous sodium sulfate, and then evaporated in vacuo, first
at 30 °C, and then at 70 °C for 30 min. The residue (1.9 g,
product I) was dissolved in 3.5 mL of DMSO, and then 0.76 g
∼ 130 mg. UV: λmax ) 365 nm (ε ) 14 300 M-1 cm-1), λmin
)
295 nm (ε ) 2800 M-1 cm-1). 1H NMR chemical shifts (d) in
DMF were as follows: 3.65 (3H, methyl), 3.94 (q, 2H,
3-methylene, J ) 3.6), 6.24 (broad, 7 amine), 6.72 (m, 1H, 6H),
6.72 (m, 1H, 8H), 7.48 (1H, 5H), and 11.9 (broad, amide).
2. 7-Amino-4-trifluoromethyl-3-carboxy-2(1H) quinolone
(III) (cs124CF3-CH2COOH). One milliliter of 1 M aqueous
NaOH was added to 100 mg of product II dissolved in 2 mL
dioxane. After 4 h incubation at 50 °C, the mixture was diluted
by the addition of 15 mL water and extracted with ether. The
product (III) was precipitated from the aqueous phase by the
addition of citric acid to pH 3-3.5, collected by centrifugation,
washed a few times with water until neutral reaction, and dried
in vacuo. Yield ∼ 70 mg.
The aqueous phase obtained after ether extraction (see
previous section), containing product III, was acidified by the
addition of citric acid to pH 3-3.5; the precipitate was collected,
washed a few times with water, dried, and then combined with
the above. Recrystallization from ethyl acetate resulted in the
1
isolation of pure product III (total yield ∼ 200 mg). H NMR
chemical shifts (d) in DMF were as follows: 3.93 (2H, 4

Ultrasensitive Detection of Nucleic Acids
Bioconjugate Chem., Vol. 21, No. 2, 2010 321
Scheme 1. Schemes for the Synthesis of Probe 1 (A) and Probe 2 (B)
methylene), 6.19 (2H broad, 7 amine), 6.71 (1H, 6H), 6.71 (1H,
8H), 7.48 (1H, 5H), 11.85 (broad, amide), and 12.75 (broad,
carboxyl).
7.2), 7.30 (t, 6H, m-ArH, J ) 7.4), 7.48 (d, 6H, o-ArH, J )
7.4), 7.48 (1H, 5H), and 11.75 (1H, broad, amide).
4. cs124CF3-CH2C(O)-NH(CH2)6NdCdS (V). 140 mg of
compound IV were dissolved in 2 mL of 90% acetic acid and
incubated at 90 °C for 15 min. After evaporation in vacuo, the
resulting residue was suspended in water and extracted with
ether to remove triphenylcarbinol. The aqueous phase was then
evaporated to dryness. The resulting residue was dissolved in
2 mL of methanol, and 80 mg of thiocarbonyldiimadazole was
then slowly added to this solution under rigorous agitation. After
10 min incubation at room temperature, the mixture was
supplemented with 100 µL trifluoroacetic acid and kept at 50
°C. Thin layer chromatographic analysis in ethylacetate/ethanol
(12:1) revealed nearly quantitative conversion of the original
compound (Rf ) 0.05) to an isothiocyanate (Rf ) 0.40). This
product was purified by column chromatography using the same
3. cs124CF3-CH2C(O)-NH(CH2)6NH-Tr (IV). 100 mg (∼0.3
mmol) of product III were dissolved in 8 mL of THF and
supplemented with 210 mg (1 mmol) of DCC. After 1 h
incubation, thin layer chromatographic analysis in ethyl acetate/
ethanol (10:1) revealed a single product (Rf ) 0.80) with intense
blue fluorescence. 450 µmol of N-trityl-1,6-diaminohexane were
added and incubation continued for another 30 min at 20 °C.
Thin layer chromatography in ethylacetate/ethanol (10:1) re-
vealed the main reaction product (Rf ) 0.70). This mixture was
diluted with 20 mL of 0.1 M aqueous Na2CO3 and extracted
with an equal volume of chloroform. The organic phase was
then collected and rinsed with 1 volume 0.2 M citric acid, dried
over anhydrous sodium sulfate, and the solvent removed by
evaporation under reduced pressure. The product was purified
by silica gel chromatography, using ethylacetate/ethanol (10:1)
1
eluent. Yield ∼ 60 mg. H NMR chemical shifts (d) in DMF
1
were as follows: 1.35 (m, 4H), 1.46 (m, 2H), 1.65 (m, 2H),
3.13 (m, 2H, J ) 7.25), 3.69 (t, 1H, J ) 7.2), 3.08 (q, 2H, J )
7.2), 3.82 (q, 2H, 3CH2-, J ) 3.7), 6.14 (s, 2H, broad, 7 amine),
6.7 (1H, 8H), 6.7 (1H, 6H), 7.48 (m, 1H, 5H), 7.76 (t, 1H, J )
5.5), and 11.77 (1H, broad, amide).
mixture as eluent. Yield ∼ 160 mg. H NMR chemical shifts
(d) in DMF were as follows: 1.45 (m, 8H), 1.65 (m, 2H), 1.78
(m, 2H), 2.03 (q, 2H, J ) 7.25), 2.41 (t, 1H, J ) 7.2), 3.08 (q,
2H, J ) 7.2), 3.82 (q, 2H, 4CH2-, J ) 4.0), 6.14 (s, 2H, broad,
7 amine), 6.7 (1H, 8H), 6.7 (1H, 6H), 7.18 (t, 3H, p-ArH, J )

322 Bioconjugate Chem., Vol. 21, No. 2, 2010
Krasnoperov et al.
5. Lanthanide Complexes of DTPA-cs124-CF3-NCS (probe
1). Thirty milligrams (0.1 mmol) of compound V were added
to a solution of 80 mg (0.3 mmol) of DTPA dianhydride in 0.8
mL of DMSO. After incubation (45 min at 50 °C), the mixture
was supplemented with 10 mL of ether, and the resulting
precipitate was spun down, washed with ether, air-dried,
dissolved in 1 mL of DMF, and mixed with 0.3 mL of water.
After incubation for 10 min at 45 °C, the mixture was diluted
with 5 mL of water and extracted with 40 mL of butanol. The
organic phase was separated and divided into four equal parts.
Each portion was mixed with 0.3 mL of a 0.1 M solution of a
lanthanide trichloride (Tb3+, europium (Eu3+), dysprosium
(Dy3+), and samarium (Sm3+)). After vigorous agitation, the
organic phase was collected and concentrated by co-evaporation
with water in vacuo at 30 °C. Analytical thin layer chromatog-
raphy, using an acetonitrile/water system (3:1) as the developing
solvent, revealed two main Ln3+ products (Rf ) 0.25 and 0.50).
The products with Rf ) 0.50 (desired compound) were purified
using preparative thin layer chromatography under the same
conditions. The fluorescent material was eluted with 50%
aqueous ethanol and was recovered as a colorless powder after
evaporation in vacuo. UV: λmax ) 347 nm (ε ) 14 800 M-1
cm-1), λmin ) 270 nm (ε ) 4700 M-1 cm-1). MS: Eu3+DTPA-
cs124-CF3-CH2C (O)-NH(CH2)6NdCdS (-H+) 950.1 (found),
950.0 (calculated). Ln3+ complexes of DTPA-cs124-CF3 were
obtained using the same protocol.
(m, 6H), 1.60 (m, 2H, e-CH2, J ) 7.2), 3.04 (m, 2H, R-CH2, J
) 7.2), 3.49 (s, 2H, 4-methylene), 3.64 (t, 2H, ꢀ-CH2, J ) 7.2),
5.75 (s, 2H, broad, 7 amine), 5.98 (s, 1H, 3H), 6.36 (d, 1H,
8H, J ) 2.4), 6.43 (dd, 1H, 6H, J1 ) 7.2, J2 ) 2.4), 7.38 (d,
1H, 5H, J ) 7.2), 8.07 (t, 1H, amide, J ) 7.2), and 11.20 (1H,
broad, amide quinolone).
4. Lanthanide Complexes of DTPA-cs124 and DTPA-cs124-
NCS (probe 2). These products were obtained and purified
essentially as described for the synthesis of analogous probe 1
compounds in section 5 above; however, the incubation time
of the corresponding isothiocyano compound (IX) with DTPA
dianhydride was 15 min at 20 °C. UV: λmax ) 341 nm (ε )
18 900 M-1 cm-1), λmin ) 308 nm (ε ) 10 000 M-1 cm-1).
MS: Tb-DTPA-cs124-NCS (-1) 888.3 (found), 888.0 (calcd);
and Eu-DTPA-cs124-NCS (-1) 882.3 (found), 882.0 (calcd).
Synthesis of Luminescent Hybridization Probes. A 10 µL
water solution containing 3 to 7 nmol of an oligonucleotide (5′
amino - CTTCGTCCACAAACACAACTCCTGAAG - 3′ Black-
hole Quencher 2), prepared according to protocols described
previously (20), was supplemented with 5 µL of Na2B4O7 (pH
10.0), and 15 µL of a 10 to 20 mM aqueous solution of
luminescent probe 1 or probe 2. After incubation for 3.5 h at
56 °C, the probe conjugated oligonucleotide was precipitated
by the addition of 200 µL of ethanol, and then collected by
centrifugation after cooling at -80 °C for 15 min. This
procedure was repeated 3 to 4 times. Finally, the residue was
dissolved in water and purified by HPLC chromatography, as
described in Supporting Information. Yield 40-80%.
Physical Methods. Excitation and emission fluorescence
spectra in a steady-state mode were recorded using a Quanta-
Master 1 (Photon Technology International) digital fluorometer
at ambient temperature. Time-resolved and gated luminescence
measurements were performed using a home-built experimental
setup (Supporting Information Figure S1). A Suprasil fluores-
cence cell filled with sample solutions was irradiated by pulsed
(ca. 15 ns) UV light from an excimer laser (351 nm, XeF).
Before passing through the cell, the laser beam was formed by
a rectangular aperture 0.5 cm × 1.0 cm (width × height).
Fluorescence from the cell collected at an angle of 90 degrees
was focused onto the entrance slit of a grating spectrograph
(SpectraPro-300i, Acton Research Corporation, diffraction grat-
ing 150 grooves/mm blazed at 500 nm) using a fused silica
lens with a focal distance of 2.5 cm. The spectrograph was
equipped with a gated intensified CCD Camera (ICCD-MAX,
Princeton Instruments) to record transient spectra. A slit width
of 0.5 mm was used for time-resolved luminescence measure-
ments, which corresponds to a spectral resolution of 5 nm. Time-
gated spectra were recorded with a spectral resolution of 0.3
nm (a slit width of 0.01 mm in combination with a pixel size
on the ICCD camera of 0.026 mm). ICCD gating, with a delay
after the laser pulse, was used to determine the temporal
behavior of the transient fluorescence. For measurements of
luminescence lifetimes, the light was diverted to a photomul-
tiplier tube mounted on the exit slit of the spectrograph. The
PMT signal was preamplified and averaged, using a digital
storage oscilloscope (LeCroy 9310A). High-resolution spectra
were recorded with a time delay of 1 µs and a gate width of 1
ms for probe 1 chelated with Eu and Sm and for probe 2 chelated
with Tb and Dy.
Probe 2 (Scheme 1B). 1. 7-Amino-4-carboethoxymethyl-2(1H)
quinolone (VII). A suspension of 1.36 g (10 mmol) of ZnCl2 in
5 mL of DMSO was supplemented with 1.08 g (10 mmol) of
1,3-phelylenediamine and 2.02 g (10 mmol) of diethyl-1,3-
acetonedicarboxylate. The mixture was kept at 95 to 100 °C
for 24 h. Thin layer chromatography in chloroform/ethanol
(10:1) detected one main fluorescent product (Rf ) 0.35). This
mixture was diluted with 8 mL of ethanol, poured into 150 mL
of ice-cold 0.1 M citric acid, and left for 3 h at 4 °C. The residue
was filtered and successively washed with water (2 × 10 mL),
and with hot acetonitrile (2 × 5 mL), and then dried in vacuo.
1
Yield ∼ 1.4 g (60%). H NMR chemical shifts (d) in DMSO
were as follows: 1.17 (t, 3H, -OCH2CH3, J ) 7.2), 3.76 (s, 2H,
3-methylene), 4.06 (q, 2H, -OCH2CH3, J ) 7.2), 5.81 (2H,
broad, 7 amino), 6.01 (s, 1H, 3H), 6.37 (d, 1H, 8H, J ) 2.4),
6.43 (dd, 1H, 6H, J1 ) 7.2, J2 ) 2.4), 7.25 (d, 1H, 5H, J )
7.2), and 11.28 (1H, broad, amide).
2. 7-Amino-4-carboxamido(6-aminohexyl)methyl-2(1H) qui-
nolone (VIII). Premelted 1,6-diaminohexane (2 g, 17 mmol) was
mixed with 7-amino-4-carboethoxymethyl-2(1H) quinolone (0.5
g, 2 mmol). After incubation (15 h, 90 °C), the mixture was
poured into 30 mL of water. The precipitate was washed with
water (3 × 30 mL) and ethyl acetate (3 × 20 mL), and then
stirred with hot methanol (50 mL), filtered, and the filtrate
evaporated to dryness in vacuo. The product appeared as light-
1
brown crystals. Yield ∼ 0.5 g. H NMR chemical shifts (d) in
DMSO were as follows: 1.2-1.4 (m, 10H), 3.04 (q, 2H, R-CH2,
J ) 7.2), 3.49 (s, 2H, 4-methylene), 5.75 (2H, broad, 7 amino),
5.98 (s, 1H, 3H), 6.36 (d, 1H, 8H, J ) 2.4), 6.43 (dd, 1H, 6H,
J1 ) 7.2, J2 ) 2.4), 7.38 (d, 1H, 5H, J ) 7.2), 8.07 (t, 1H,
amide, J ) 7.2), and 11.3 (1H, broad, amide quinolone).
3. 7-Amino-4-carboxamido (6-isothiocyanohexyl) methyl-2
(1H) quinolone (IX). 44 mg (0.22 mmol) of 1,1′-thiocarbonyl-
diimidazole was added to a solution of 63 mg (0.2 mmol) of
product VIII dissolved in 4 mL methanol. After 5 min, this
mixture was supplemented with 10 µL of TFA and incubated
for 40 min at 50 °C. The solvent was removed by evaporation
in vacuo, and the product was then washed with water and
purified by column chromatography on silica gel using a
chloroform/ethanol mixture (4:1) as eluent. Yield ∼ 40 mg. 1H
NMR chemical shifts (d) in DMSO were as follows: 1.2-1.5
Steady-State Fluorescence Measurements. Hybridization
experiments of the lanthanide-based molecular beacon with its
complementary target DNA (5′ TTAGGAGTTGTGTTTGTG-
GACTT 3′) were performed in a measuring cell (150 µL) in a
hybridization buffer containing 50 mM KCl, 3 mM MgCl2, and
10 mM Tris-HCl (pH 8.0). The concentrations of the molecular
beacon and the cDNA oligonucleotide (target) were 300 nM

Ultrasensitive Detection of Nucleic Acids
Bioconjugate Chem., Vol. 21, No. 2, 2010 323
and 1000 nM, respectively. Water-based or deuterium oxide-
based solutions were used.
Time-Resolved Luminescence Measurements. Protocol
A. Ten microliters of various concentrations (0-100 nM) of
target DNA (see previous section) was added to 3 mL of a 1
nM solution of molecular beacons in hybridization buffer (see
previous section), and was then transferred to a measuring cell.
The luminescence of the sample was measured at different time
intervals.
Protocol B. Different concentrations (0-3 nM) of DNA target
were added to 3 mL of 10 pM molecular beacon hybridization
buffer in a glass tube, and this solution was left for 3 days at
room temperature. The solution was then transferred to the
measuring cell. The glass tube was washed with 1 mL of the
same hybridization buffer containing 30% ethanol, and this
solution was also transferred to the measuring cell. The resulting
luminescent signal was then measured as described above.
RESULTS
Synthesis and Properties of Cross-Linkable Lanthanide
Chelates. The structure of the synthesized luminescent lan-
thanide probes is shown schematically in Chart 1. These probes
are derivatives of previously described cs124 and cs124-CF3
fluorophores (10-17). For the synthesis of these compounds,
we used an approach based on the condensation of 1,3-
phenylenediamine with an ester of an acetoacetic acid derivative.
Scheme 1A presents the synthetic strategy developed for probe
1. Alkylation of trifluoroacetoacetate by methylbromoacetate
in the presence of a proton acceptor produced trifluoroacetyl-
methylethylsuccinate (compound I). Reaction of the latter with
1,3-phenylenediamine resulted in fluorescent quinolone deriva-
tive II, which was converted to the corresponding carboxyl
derivative III by saponification. We intended to introduce the
amino function in this compound by treatment with DCC in
the presence of 4-nitrophenol and subsequent reaction of
nitrophenyl ester with aliphatic diamine. Surprisingly, incubation
with DCC and 4-nitrophenol yielded a product that did not
contain 4-nitrophenol. The ultraviolet spectrum of this product
differed significantly from that of compound III. In aqueous
medium, this product converted into III with a half-time of about
30 min at 20 °C. Incubation of this product with aliphatic amines
resulted in acylation of the amino groups. Most likely, this
reactive intermediate emerged as a result of intramolecular
acylation of the amide oxygen of III by activated carboxylate.
Incubation of this compound with monotritylated 1,6-diamino-
hexane yielded the desired compound IV. After deprotection,
the resulting aminoalkyl derivative was converted to isothio-
cyanate (compound V) by treatment with thiocarbonyldiimida-
zole and trifluoroacetic acid. Under these conditions, the
aromatic amino group of the compound remains intact. Acy-
lation of compound V by DTPA anhydride in anhydrous
medium, followed by hydrolysis of the second anhydride group,
resulted in compound VI (probe 1), which was separated from
the excess of DTPA by partitioning in butanol/water. Further
addition of aqueous lanthanide trichloride to the butanol extract
led to complexes between the lanthanides and the probes. The
lanthanide complexes were analyzed and purified by thin layer
chromatography in acetonitrile:water mixtures that proved to
be highly efficient (see Figure 1).
Figure 1. Image recorded by ultraviolet excitation of ligand antenna
compounds (-Me) and their lanthanide complexes (Tb and Eu), after
separation by thin layer chromatography.
Figure 2. UV absorption spectra of synthesized reactive luminescent
probes and reference compounds used in this study.
of the reaction temperature. Reaction of this product with a
diamine (added in a few-fold excess to avoid cross-linked
products) at high temperature resulted in the formation of
aminoalkyl derivative VIII, with nearly quantitative yield.
Cross-linkable DTPA derivative (X, probe 2) was obtained in
the same manner as probe 1.
Absorption Spectra. Absorption spectra of the synthesized
cs124 and cs124-CF3 derivatives are shown in Figure 2. They
were nearly identical to published spectra for analogous
compounds (10-17). A small red shift of 6 nm was observed
for probe 1 (comparing to the reference compound cs124-CF3)
and probe 2 (comparing to the reference compound cs124). The
molar extinction coefficients for DTPA-cs124 and DTPA-cs124-
CF3 were determined by direct comparison of the absorption
spectra of the original compounds (εmax ) 18 900 M-1 cm-1 at
341 nm for cs124 and εmax ) 14 500 M-1 cm-1 at 360 nm for
cs124-CF3) to their acylated derivatives, which was achieved
by monitoring spectral changes in the reaction mixture during
the course of the reaction (see Supporting Information Figures
S2A and S2B). The presence of isosbestic points in both cases
was indicative of the conversion of the original compounds to
For the synthesis of probe 2, we developed the strategy shown
in Scheme 1B. The first step of this synthesis was based on a
published protocol (13) that includes incubation of m-phe-
nylenediamine with diethyl 1,3-acetonedicarboxylate at high
temperature to yield quinolone derivative VII. Compared to
published protocols, a dramatic improvement in the yield of
the final product (from <1% to ca. 60%) was achieved by using
a mild Lewis acid (ZnCl2) as a catalyst, which allowed lowering

324 Bioconjugate Chem., Vol. 21, No. 2, 2010
Krasnoperov et al.
brightness of the lanthanide chelates in different ways. For
cs124-CF3-based antennae, a significant decrease in brightness
was observed for Tb3+ chelates (ca. 20-fold) and for Dy3+
chelates (>30-fold), while the emission of Eu3+ and Sm3+
chelates was not significantly affected. A similar effect of
substitution was previously observed for the analogous Tb-cs124
chelates (13). In the case of probe 2, the substitution of a cross-
linking group for a methyl group in position 4 did not
significantly alter the brightness of all lanthanide complexes.
Moreover, a detectable increase in the brightness for Eu (1.5-
fold), Dy (1.7-fold), and Sm (1.6-fold) complexes was observed.
This is consistent with the results (13) previously obtained for
the analogous Tb and Eu derivatives of cs124 containing a
carboxymethyl group at position 4. Surprisingly, significant
luminescence was detected for Tb3+-DTPA-cs124-CF3 chelates,
which were previously reported to be nonluminescent. We do
not know the reason for this discrepancy. As seen in Table 1,
comparing the emission of probe 1 and probe 2, probe 1 gives
brighter complexes with Eu and Sm, while probe 2 is optimal
for Tb and Dy. Time-resolved measurements indicated that there
is a single-exponential decay mode for the luminescent signal
from probe 1 chelates and from probe 2 chelates (not shown),
which is indicative of the homogeneity of the complexes.
Effect of Heavy Water on Lanthanide Chelate Emission. The
quantum yield of the excited lanthanide ion (defined as the
probability of the excited state emitting a photon) in the an-
tenna-chelate complex depends strongly on the number of
coordinated water molecules (21), due to nonradiative dissipation
of the energy of the excited state through the vibration of O-H
bonds. This process does not occur with heavy water due to
the different frequency of O-D bond vibration. This effect
accounts for the enhanced brightness of lanthanide luminescence
in heavy water. Indeed, as seen from Table 1 for DTPA ligands
in D2O, the brightness of the Tb3+ chelates was 1.3- to 1.5-fold
higher than in H2O-based solutions. As expected, the effect was
more pronounced for DTPA-Eu3+ chelates (∼3- to 3.8-fold),
as well as for Dy3+ and Sm3+ complexes (∼ 4- to 6-fold). The
number of coordinated water molecules in Tb3+ and Eu3+
complexes can be calculated from the luminescence lifetime in
water and in deuterium oxide-based solutions (22). For our
probes, the number of coordinated water molecules was close
to unity (see Table 1), which is in agreement with the results
reported for similar compounds. The same is expected for Dy3+
and Sm3+ chelates, since they have analogous coordination
chemistry.
Figure 3. (A) Luminescence of 50 µM solutions of probe 2 (no Me)
and its lanthanide complexes: terbium (Tb), europium (Eu), dysprosium
(Dy), samarium (Sm); in heavy water, excited at 308 nm. (B)
Normalized time-resolved emission of probe 1 Eu and Sm chelates
and of probe 2 Tb and Dy chelates.
a single reaction product. Indeed, chromatographic analysis
confirmed the formation of single acylation products in both
cases. Thus, values of εmax ) 18 200 M-1 cm-1 at 328 nm and
εmax ) 14 800 M-1 cm-1 at 341 nm were obtained for DTPA-
acylated derivatives of cs124 and cs124-CF3, respectively.
Essentially the same values were obtained for probes 1 and 2
(data not shown). A significant difference in the absorption
spectra of probes 1 and 2 (18 nm) allows selective excitation
by common sources (at 351 nm by excimer XeF laser for probe
2, and at 365 nm by mercury UV lamp for probe 2). Such
selective excitation is important for applications relying on
simultaneous monitoring of two independent processes in the
same sample.
Emission Spectroscopy of Lanthanide Complexes. The ease
of observing the luminescence of these lanthanide complexes
is illustrated in Figure 3A for probe 2. All complexes displayed
the narrow emission spectrum typical of luminescent lanthanide
chelates (Figure 3B). Table 1 shows the luminescence intensities
of the synthesized reactive lanthanide chelates, as well as for
the intensities obtained for the reference compounds, cs124 and
cs124-CF3 described earlier (11, 17). The process of antenna-
mediated lanthanide emission includes the transfer of energy
from the antenna fluorophore to the coordinated metal and the
subsequent emission of photons by the excited lanthanide. The
first step is the most crucial part of the process, because even
slight modifications of chromophore-antenna structure dramati-
cally affect lanthanide luminescence (13). In this work, we
explored a synthetic approach that allows the introduction of a
cross-linking group in position 3 (probe 1) or position 4 (probe
2) of quinolone-based antenna fluorophores. Comparison with
reference fluorophores (with a non-substituted quinolone at
position 3 or a methyl-substituted quinolone at position 4)
demonstrates that the structural modification affected the
Effect of EDTA on the Rate of Lanthanide Chelate Decay.
The ability to retain the chelated metal ion is an important
characteristic of lanthanide probes. This property is especially
crucial for intracellular applications, due to the abundance of
metal scavengers in living cells (such as free amino acids, amino
acid residues in proteins, nucleoside triphosphates, nucleic acids,
etc.). To this end, we investigated the decay rates of the
lanthanide chelates used in our probes and in those probes
described previously (15). Reaction of cs124 with excess of
DTPA dianhydride yields a primary adduct possessing a
preserved acylation function. In previous studies, this function
was used to attach the cross-linking group to the chelate
construct by subsequent treatment with diamine, which yielded
aminoalkylamide derivatives whose amino group was converted
to amine reactive isothiocyanates. This modification of the
chelating group is likely to weaken the retention of metal. To
this end, as a model, we synthesized analogous, but nonreactive,
compounds containing a butylamide group. In accordance with
expectations, the lanthanide complexes were about 10 times less
stable (comparing to those possessing nonmodified chelating
groups) when challenged with EDTA (see Supporting Informa-
tion Figure S3).

Ultrasensitive Detection of Nucleic Acids
Bioconjugate Chem., Vol. 21, No. 2, 2010 325
Table 1. Emission and Relative Brightness of Lanthanide Chelates under Various Conditions
relative
number of
emission in H2O, emission in D2O, relative brightness brightness in life time in H2O life time in D2O coordinated H2O
compound
counts
counts
D2O/H2O
H2O (%)
(ms)
(ms)
molecules
Dysprosium (Dy3+) Complexes (emission at 482 nm)
b
DTPA-cs124-CF3
555
-
2720
4550
917
-
10522
13000
1.75
-
3.87
2.86
20
-
100
167
0.0023
-
0.011
0.009
0.0045
-
0.033
0.027
DTPA-cs124-CF3-NCS (probe 1)
DTPA-cs124b
-
-
DTPA-cs124-NCS (probe 2)
Terbium (Tb3+) Complexes (emission at 545 nm)
b
DTPA-cs124-CF3
12,200
833
169000
137000
14170
790
260000
190000
1.16
0.95
1.53
1.40
7.2
0.5
100
81
0.2
0.2
-
-
DTPA-cs124-CF3-NCS (probe 1)
-
-
DTPA-cs124b
1.5 (1.55a)
1.2
2.3 (2.63a)
1.7
0.97 (1.1a)
1.03
DTPA-cs124-NCS (probe 2)
Samarium (Sm3+) Complexes (emission at 598 nm)
b
DTPA-cs124-CF3
192
230
114
180
764
900
513
3.98
3.91
4.50
6.60
168
200
100
158
0.0080
0.0092
0.0082
0.0082
0.036
0.042
0.023
0.034
DTPA-cs124-CF3-NCS (probe 1)
DTPA-cs124b
-
-
DTPA-cs124-NCS (probe 2)
1010
Europium (Eu3+) Complexes (emission at 615 nm)
b
DTPA-cs124-CF3
10350
9450
5050
7300
41000
35000
22000
25000
3.96
3.70
4.36
3.42
205
187
100
145
0.5
0.5
1.9
1.7
1.54
DTPA-cs124-CF3-NCS (probe 1)
1.47
DTPA-cs124b
0.6 (0.62a)
0.6
1.6 (2.42a)
2.0
1.1 (1.26a)
1.19
DTPA-cs124-NCS (probe 2)
a Determined in ref 13. b Model compounds.
Chemical Reactivity of Synthesized Luminescent Probes. The
chemical reactivity of the synthesized probes was evaluated first
in reactions with aliphatic diamines and with cysteine, because
the resulting reaction products (corresponding thioureas and
thiocarbamates) can easily be identified by thin layer chroma-
tography, due to the strong retardation effect. Under the
incubation conditions that were used, a nearly quantitative
conversion of the probes to the corresponding reaction products
was observed, either immediately upon mixing (with 0.1 M
cysteine) or after 2 to 3 h at 56 °C (with 10 mM diamine),
suggesting that the isothiocyanate groups in the probes survived
purification. At the same time, nonreactive control chelates
(Ln3+-DTPA-cs124 and Ln3+-DTPA-cs124-CF3) did not change
their mobility after incubation under the same conditions (see
Supporting Information).
Synthesis of Luminescent-Based Molecular Beacon Probes.
With conventional strategies, the nucleic acids are first coupled
with metal-free probe derivatives, followed by the addition of
a lanthanide to the probe conjugated products, which is not
convenient. To this end, we obtained metal-chelated, ready-to-
use luminescent tags. This was especially important for probe
1, since a metal-free compound is not stable.
For the attachment of the luminescent tags to the oligonucle-
otides, we obtained aliphatic isothiocyanate-reactive probes, as
opposed to their more reactive aromatic counterparts that are
typically used for modification of biopolymers. Partially, this
choice was due to a synthetic strategy that required survival of
the reactive cross-linking group during acylation by DTPA
dianhydride (see Scheme 1) and during subsequent purification.
Also, a closely positioned aromatic moiety of isothiocyanate
could affect the energy transferred to the lanthanide by the
occurrence of stacking interactions with antenna fluorophores.
Under optimized reaction conditions, we were able to achieve
a 70% coupling efficiency with amine-containing oligonucle-
otide constructs. The resulting hybridization probes were purified
by ethanol precipitation, followed by size-exclusion chroma-
tography and HPLC. Comparison of the absorption spectra of
3′ BHQ-2 labeled oligonucleotides, with and without probe 2
attached to their 5′ terminal (Figure 4B), revealed additional
absorption in the range 300-350 nm due to the presence of the
luminescent label. The survival of the lanthanide chelates during
all of these operations is indicative of the extraordinarily high
chemical stability of these probes.
Steady-State Fluorescence Measurements of Molecular Bea-
con Probes. Molecular beacon probes are nucleic acid hybrid-
ization probes that fluoresce when they bind to a target DNA
or RNA sequence (19). When they are free in solution and not
hybridized to a target nucleic acid, they remain nonfluorescent.
Figure 4A illustrates the principle of molecular beacon probes.
The probes are single-stranded oligonucleotides that form a
stem-and-loop structure. The loop portion of the oligonucleotide
is a probe sequence that is complementary to a target sequence
in a nucleic acid. The probe sequence is embedded between
two “arm” sequences, which are complementary to each other.
Under assay conditions, the arms bind to each other to form a
double-helical stem hybrid that encloses the probe sequence,
forming a hairpin structure. A reporter fluorophore is attached
to one end of the oligonucleotide and a nonfluorescent quencher
is attached to the other end of the oligonucleotide. The stem
hybrid brings the fluorophore and quencher in close proximity,
allowing energy from the fluorophore to be transferred directly
to the quencher through contact quenching. At assay tempera-
tures, when the probe encounters a target nucleic acid, it forms
a relatively rigid probe-target hybrid that is longer and more
stable than the stem hybrid. Consequently, the molecular beacon
probe undergoes a conformational reorganization that forces the
stem hybrid to dissociate, and results in the separation of the
fluorophore and the quencher, restoring fluorescence.
Currently, most molecular beacon probes are labeled with
organic fluorophore labels (23). To increase the sensitivity of
detection, we explored the performance of lanthanide-based
molecular beacons by using the novel luminescent probes
described in this study, instead of probes possessing conven-
tional fluorophores. An example is shown in Figure 4C. In this
case, we used a europium complex of probe 1 as the lumino-
phore for molecular beacon. As expected, the addition of a
cDNA target resulted in the development of a signal that is
distinguished by the narrow emission peaks that occur in
lanthanide luminescence. Remarkably, the ratio of the intensity
of the luminescence signal to the intensity of the background
fluorescence of the molecular beacon (which is due to the
fluorescence of the molecular beacon after subtraction of the
background fluorescence of the medium) was >400, which is
significantly higher than the signal-to-background ratio typically
obtained for fluorophore-based hybridization probes, including
molecular beacons (23). This higher signal-to-background ratio

326 Bioconjugate Chem., Vol. 21, No. 2, 2010
Krasnoperov et al.
Figure 5. Time-resolved detection of a cDNA oligonucleotide target
using a Eu-probe 1-based luminescent molecular beacon. (A) Emission
intensity of a molecular beacon (1 nM) in the presence of different
concentrations of target. (B) End-point emission detection of an
oligonucleotide target at different concentrations in the presence of a
10 pM molecular beacon.
Figure 4. Detection of cDNA oligonucleotide targets using luminescence-
based molecular beacons labeled by probe 1 Eu chelate in a steady-
state mode. (A) Principle of detection. (B) Normalized absorption
spectra of 3′ BHQ-2 labeled oligonucleotides, with (solid line) and
without (dotted line) probe 2 conjugated to their 5′ terminal. (C)
Luminescence emission spectra of the hybridization buffer (dashed line),
the molecular beacon alone (dotted line), and the molecular beacon
after hybridization to a complementary oligonucleotide (solid line).
detection limits as low as 0.5-1 pM (Figure 5B), which is about
50- to 100-fold more sensitive than the results that are obtained
in the same system using conventional fluorescein-based mo-
lecular beacons. Moreover, these detection limits are better than
those reported previously for other lanthanide-based hybridiza-
tion probes.
DISCUSSION
is due to both the suppression of the fluorescence of the antenna
and the luminescence of the lanthanide in the “closed” form of
the molecular beacon by the quencher. As expected, the signal
that occurs upon hybridization of the probe to its target is
significantly brighter in heavy water-based solutions.
The unique photon emission properties of lanthanide-based
probes render them suitable for a wide variety of applications
that require ultrasensitive detection of biomolecules. Progress
in this field depends on the availability of efficient probes. The
mechanism of energy pathways in luminescent lanthanide
chelates is not fully understood, leaving much room for
improvements in their applications as labels for probes. Even
though the first probes of this class were synthesized more than
three decades ago and are not very efficient, many of them
continue to be used for commercial applications. The develop-
ment of more efficient probes is highly desirable, because new,
more challenging applications have arisen (e.g., for the detection
of rare pathogens in environmental samples, and for the
detection of single molecules in cells).
Time-Resolved Luminescence Measurement of Molecular
Beacons Probes. Figure 5A shows the time-course for the
development of the luminescence signal that is detected in the
time-resolved mode from hybridization mixtures that contain
probe 1-based molecular beacons and various concentrations
of complementary target DNA. The results demonstrate that,
when 1 nM hybridization probe is present, subnanomolar
concentrations of the target can be detected after only 10 min
of incubation at ambient temperature. As seen from the time-
course curves at low concentrations of the target, the hybridiza-
tion rate decreases significantly, suggesting that the sensitivity
of the detection can be improved by increasing the incubation
time. Indeed, by lowering the concentration of the hybridization
probe to 10 pM (to reduce background emission), and by
prolonging the incubation time, we were able to achieve
In the present studies, we have taken advantage of new
synthetic strategies that enable the preparation of highly
luminescent probes with high yield. The resulting probes are
ready-to-use, since they contain prebound lanthanides, unlike
the lanthanide-labeled probes utilized in earlier applications, in

Ultrasensitive Detection of Nucleic Acids
Bioconjugate Chem., Vol. 21, No. 2, 2010 327
strong circularly polarized luminescence. J. Am. Chem. Soc. 129,
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which the biomolecule of interest was first modified with metal-
free chelates, followed by the addition of a lanthanide (10, 15).
The new compounds that we describe were tested in the form
of molecular beacons, which are widely utilized nucleic acid
hybridization probes. Our experiments revealed a higher sen-
sitivity of detection than can be achieved with conventional
fluorescence-based molecular beacons. Moreover, the detection
sensitivity was 10 to 60 times better than previously reported
for other lanthanide-based hybridization probes (24, 25). Also,
the brightness of the probes is significantly increased in heavy
water, suggesting the use of this medium to increase the
sensitivity of detection. Because of the superior properties, these
new compounds could also be used as luminescent labels in
other biopolymers, such as proteins and polysaccharides, as well
as labels for small compounds, such as drugs and cellular
metabolites.
(14) Ge, P., and Selvin, P. R. (2008) New 9- or 10-dentate
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ACKNOWLEDGMENT
(15) Li, M., and Selvin, P. R. (1997) Amine-reactive forms of a
luminescent diethylenetriaminepentaacetic acid chelate of terbium
and europium: attachment to DNA and energy transfer measure-
ments. Bioconjugate Chem. 8, 127–32.
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transfer using a terbium chelate: improvements on fluorescence
energy transfer. Proc. Natl. Acad. Sci. U.S.A. 91, 10024–8.
(17) Selvin, P. R., Rana, T. M., and Hearst, J. E. (1994)
Luminescence Resonance Energy Transfer. J. Am. Chem. Soc.
116, 6029–6030.
(18) Heyduk, E., and Heyduk, T. (1997) Thiol-reactive, luminescent
Europium chelates: luminescence probes for resonance energy
transfer distance measurements in biomolecules. Anal. Biochem.
248, 216–27.
Authors are grateful to Fred Russell Kramer and Sanjay Tyagi
for helpful discussion and comments. This program was
supported by NIH grant R01 GM-30717-21 for AM and by NIH
grant RO1 MH-079197 for SM.
Supporting Information Available: Detailed protocols,
including protocols for determining the luminescent and optical
properties of the synthesized compounds, studies of their
chemical reactivity and stability, as well as a description for
purification of the molecular beacon hybridization probes.This
material is available free of charge via the Internet at http://
pubs.acs.org.
(19) Tyagi, S., and Kramer, F. R. (1996) Molecular beacons: probes
that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–8.
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(2000) Molecular beacons: hybridization probes for detection of
nucleic acids in homogeneous solutions, in NonradioactiVe
Analysis of Biomolecules, 2nd ed. (Kessler, C., Ed.) pp 606-
16, Springer Verlag, Berlin, Germany.
(21) Li, M., and Selvin, P. R. (1995) Luminescent polyaminocar-
boxylate chelates of terbium and europium: The effect of chelate
structure. J. Am. Chem. Soc. 117, 8132–8138.
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