An investigation on the analytical potential of polymerized liposomes bound to lanthanide ions for protein analysis

Article abstract of DOI:10.1021/ja048963b

We present a promising approach to protein sensing based on Eu³⁺ ions incorporated into polymerized liposomes. The sensitization of Eu ³⁺ is accomplished with 5-aminosalicylic acid, which provides energy transfer for a stable referen

Full text of DOI:10.1021/ja048963b

Published on Web 08/05/2004
An Investigation on the Analytical Potential of Polymerized
Liposomes Bound to Lanthanide Ions for Protein Analysis
Marina Santos,† Bidhan C. Roy,‡ He´ctor Goicoechea,‡,§ Andres D. Campiglia,*,† and
Sanku Mallik‡
Contribution from the Department of Chemistry, P.O. Box 25000, UniVersity of Central Florida,
Orlando, Florida 32816-2366, and Department of Chemistry and Molecular Biology,
North Dakota State UniVersity, Fargo, North Dakota 58105
Received February 24, 2004; E-mail: acampigl@mail.ucf.edu
Abstract: We present a promising approach to protein sensing based on Eu3+ ions incorporated into
polymerized liposomes. The sensitization of Eu3+ is accomplished with 5-aminosalicylic acid, which provides
energy transfer for a stable reference signal and a wide wavelength excitation range free from protein
interference. The lipophilic character of polymerized liposomes provides the appropriate platform for protein
interaction with the lanthanide ion. Quantitative analysis is based on the linear relationship between the
luminescence signal of Eu3+ and protein concentration. Because no spectral shift of the lanthanide
luminescence is observed upon protein interaction, qualitative analysis is based on the luminescence lifetime
of polymerized liposomes. This parameter, which changes significantly upon protein-liposome interaction,
follows a well-behaved single-exponential decay that might be useful for protein identification.
aggregates.10 Attempts have been made to correct the main
disadvantage of the Bradford assay, i.e., the considerable
Introduction
Development of sensing schemes capable of recognizing
specific proteins in complex biological matrixes remains an
analytical challenge.1-3 Since 1922, when Wu proposed the use
of the Folin phenol reagent to determinate protein concentration,4
traditional approaches estimate total protein content relying on
proteins binding to organic dyes with strong absorption in the
ultraviolet and visible regions of the spectrum. The wide scope
and good reproducibility of the Lowry protein assay5 have made
it the method generally applied for this purpose. Because of its
speed of analysis and sensitivity, the binding assay reported by
Bradford6 has also become a popular alternative among re-
searchers. Despite its popularity, the Bradford procedure presents
significant disadvantages. These include different binding stoi-
chiometry between the dye (Coomassie brilliant blue G-250)
and different proteins,7,8 nonlinearity of color yield versus total
protein content,9 and incorrect quantitation of denaturated protein
underestimation of protein content of membrane-containing
fractions, which rely on sample pretreatment with membrane-
disrupting agents such as NaOH11 or detergents (Triton X-100).12
These approaches, however, do not address an inherent limita-
tion of the assay, which is the measurement of absorption in
the ultraviolet (UV) and visible (vis) ranges of the spectrum.
Spectroscopic measurements in the UV-vis are prone to strong
matrix interference. Absorption and fluorescence from con-
comitants can certainly deteriorate limits of detection, reproduc-
ibility, and accuracy of analysis.
Recent trends provide higher sensitivity by using fluorescent
dyes optically active in the near-infrared, a wavelength region
with relatively lower spectral interference from biological
matrixes.13,14 Another sensitive approach is based on the
luminescence of lanthanide ions, particularly Eu3+ and Tb3+
.
Their long-lived luminescence is a good match to time-resolved
techniques, which discriminate against short-lived background
fluorescence and scattered excitation light. Because their emis-
sion involves one of the shielded f-level electrons, their
luminescence is less sensitive to oxygen quenching than
traditional fluorescent dyes.15-17
† University of Central Florida.
‡ North Dakota State University.
§ Current address: Ca´tedra de Qu´ımica Anal´ıtica I, Facultad de Bio-
qu´ımica y Ciencias Biolo´gicas, Universidad Nacional del Litoral, Ciudad
Universitaria, CC 242-S3000, Santa Fe, Argentina.
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10738
J. AM. CHEM. SOC. 2004, 126, 10738-10745
10.1021/ja048963b CCC: $27.50 © 2004 American Chemical Society

Polymerized Liposomes Bound to Lanthanide Ions
A R T I C L E S
Offsetting these advantages is the fact that lanthanide’s
luminescence is quite weak as a result of low molar extinction
coefficients, particularly in aqueous solvents. Water molecules
strongly bind to the lanthanide ion and quench its luminescence
via weak vibronic coupling with the vibrational states of the
O-H oscillators.18 Significant enhancements for analytical use
are often achieved with chelating agents that remove water
molecules from the lanthanide’s primary coordination sphere.
The presence of a chelating agent also provides ample op-
portunity for covalent attachment of a “sensitizer” (or “antenna”)
to the coordination sphere of the lanthanide ion. Sensitizers are
typically organic molecules that strongly absorb and transfer
excitation energy to the metal ion, thereby overcoming the
inherently weak absorption of lanthanide ions.19 Because the
luminescence lifetimes of lanthanide complexes remain in the
microsecond to millisecond time domain, time-discrimination
of fluorescence background is still possible.
In this article, we introduce a promising approach to protein
sensing based on Eu3+ ions incorporated into polymerized
liposomes. Polymerized liposomes are spherically closed lipid
bilayers with aqueous interior that offer an adequate platform
for protein interaction.20 Unlike unpolymerized vesicles, proteins
cannot insert into the lipid bilayer of polymerized liposomes.
Instead, they interact with the outer lipid layer of the vesicle
via metal-ligand21,22 and receptor-ligand23,24 interactions.
Because polymerized liposomes are appreciably more stable than
their nonpolymerized counterparts, they provide more robust
platforms for protein sensing. Lanthanide ions incorporated into
polymerized liposomes have been used as magnetic resonance
contrast agents,25,26 but their potential for protein determination
has not been explored yet. Similarly, protein sensing via
polymerized liposomes has been reported, but their sensing
ability has been based on the fluorescence properties of organic
dyes.27,28 Our approach takes advantage of time-resolved
lanthanide luminescence to provide a sensitive sensing scheme
with potential applications in quantitative and qualitative analysis
of target proteins.
to primary and secondary inner filter effects. Keeping this
possibility in mind, we selected 5-aminosalycilic acid (5As) as
the sensitizer. 5As provides the luminescence enhancement
needed for reproducible Eu3+ signal intensity and a wide
wavelength excitation range free from matrix interference.
Ethylenediaminetetraacetic acid (EDTA) was chosen as the
chelating agent because it forms a tightly bound complex with
Eu3+ (binding constant being reported as ca. 1015 at pH 7).29
This fact ensures the physical integrity of the probe in the
presence of potentially competing ions and/or proteins.
Syntheses of the Complexes. Reports on polymerized
liposomes capable of complexing lanthanide ions are relatively
few. To the extent of our literature search, only one article
previous to our work30,31 reports on a polymerized liposome
containing Gd3+ for magnetic resonance imaging use.25,26,32 The
syntheses of 5As-EDTA-Eu3+ and the polymerizable lipid
incorporating this complex as the headgroup (lipid 1.Eu3+) are
shown in Scheme 1. The carboxylic acid group of EDTA-
triethyl ester33 was activated with NHS and DCC; the resultant
active ester was combined with 5As to afford the amide-ester
4. The free acid group of compound 4 was then coupled with
the reported polymerizable amine 531 to yield the lipid 1 in
protected form. The ester groups were hydrolyzed with LiOH;
lowering the pH of the reaction mixture to 3.0 (with HCl)
precipitated the lipid as a white solid. Small and giant polym-
erized liposomes from lipid 1.Eu3+ (10 wt %, 90% polymer-
izable phosphocholine PC1) were prepared following literature
procedures.34-37 The giant liposomes were found to precipitate
soon after polymerization; hence, these liposomes were not
investigated any further.
Spectral Characteristics of the Polymerized Liposomes.
Figure 1A shows the steady state (SS) excitation and emission
spectrum of EDTA-Eu3+ at neutral pH (25mM HEPES buffer).
Luminescence excitation at 395 nm promotes five characteristic
bands resulting from the electronic transitions that occur from
the 5D0 to the 7F manifold. The most intense bands correspond
5
5
to the D0-7F1 (593 nm) and D0-7F2 (616 nm) transitions.
The peaks at 580 nm (5D0-7F0), 651 nm (5D0-7F3), and 698
nm (5D0-7F4) result from forbidden transitions and, therefore,
are relatively weak. Figure 1B shows the SS excitation and
fluorescence spectra of 5As. The maximum excitation at 326
nm reduces the possibility of primary inner filter effects from
proteins. Because its fluorescence overlaps with the 466 nm
excitation peak of EDTA-Eu3+, the possibility of energy
transfer exists.
Results
Optical spectroscopy has been extensively applied to the study
of protein structure. Strong protein absorption and fluorescence
emission usually occur below 300 and 450 nm, respectively.
Spectral bands are fairly broad and typical full-widths at half-
maximum may vary from 45 to 65 nm. As previously men-
tioned, the spectral region between 200 and 450 nm is
undesirable for bioanalytical work as the assay may be prone
Figure 2A shows the SS excitation and emission spectra of
the complex (5As-EDTA-Eu3+). The broad excitation and
emission bands are attributed to the presence of the antenna.
The relatively weak luminescence from Eu3+ is overwhelmed
by the strong fluorescence of 5As, and its contribution to the
(16) Bemquerer, M. P.; Bloch, C.; Brito, H. F.; Teotonio, E. E. S.; Miranda, M.
T. M. J. Inorg. Biochem. 2002, 9, 363.
(17) Franz, K. J.; Nitz, M.; Imperiali, B. ChemBioChem 2003, 4, 265.
(18) Wu, S. L.; Horrocks, W. D., Jr. Anal. Chem. 1996, 68, 394.
(19) Sabbatini, N.; Guardigli, M.; Lehn, J.-M Coord. Chem. ReV. 1993, 123,
201.
(20) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: New
York, 1993.
(21) Megli, F. M.; Selvaggi, M.; Huber, R. Biochemistry 1998, 37, 10540.
(22) Shnek, D. R.; Pack, D. W.; Sasaki, D. Y.; Arnold, F. H. Langmuir 1994,
10, 2382.
(23) Kitano, H.; Kato, N.; Ise, N. Biotechnol. Appl. Biochem. 1991, 14, 192.
(24) Kitano, H.; Sohda, K.; Kosaka, A. Bioconjugate Chem. 1995, 6, 131.
(25) Storrs, R. W.; Tropper, F. D.; Li, H. Y.; Song, C. K.; Kuniyoshi, J. K.;
Stipkins, D. A.; Li, K. C. P.; Bednarski, M. D. J. Am. Chem. Soc. 1995,
117, 7301.
(26) Sipkings, D. A.; Cheresh, D. A.; Kazemi, M. R.; Nevin, L. M.; Bednarsky,
M. D. Nat. Med. (N.Y.) 1998, 4, 623.
(29) Parker, D.; Gareth Williams, J. A. J. Chem. Soc., Dalton Trans. 1996, 3613.
(30) Roy, B. C.; Fazal, M. A.; Arruda, A. F.; Mallik, S.; Campiglia, A. D. Org.
Lett. 2000, 2, 3067.
(31) Roy, B. C.; Santos, M.; Mallik, S.; Campiglia, A. D. J. Org. Chem. 2003,
68, 3999.
(32) Bednarski, M. D.; Lee, J. W.; Callstorm, M. R.; Li, K. C. P. Radiology
1977, 204, 263.
(33) Burks, E.; Koshti, N.; Jacobs, H.; Gopalan, A. Synlett 1998, 1285.
(34) Moscho, A.; Orwar, O.; Chiu, D. T.; Modi, B. P.; Zare, R. N. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 11443.
(35) Jaeger, D. A.; Zeng, Z. Langmuir 2003, 19, 8721.
(36) Jaeger, D. A.; Clark, T. C. Langmuir 2002, 18, 3495.
(37) Akashi, K.; Miyata, H.; Itoh, H.; Kinosita, K., Jr. Biophys. J. 1996, 71,
3242.
(27) Stora, T.; Lakey, J. H.; Vogel, H. Angew. Chem., Int. Ed. 1999, 38, 389.
(28) Malony, K. M.; Shnek, D. R.; Sasaki, D. Y.; Arnold, F. H. Chem. Biol.
1996, 3, 185.
9
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A R T I C L E S
Santos et al.
Scheme 1. Syntheses of 5As-EDTA-Eu3+ and the Polymerizable Lipid Incorporating This Complex as the Head Group (Lipid 1.Eu3+) Are
Showna
a Also shown is the structure of the polymerizable phosphocholine PC1 used as the major component of the liposomes.
SS spectrum is unnoticeable. Figure 2B depicts the SS excitation
and emission spectrum of the complex incorporated into the
liposome. Its comparison to Figure 2A shows broader excitation
and emission bands and red-shifts in both wavelength maxima.
These changes are attributed to the fluorescence contribution
from the backbone of the polymerized liposomes. Similar to
the unbound complex, the luminescence of Eu3+ does not appear
in the SS spectrum of the polymerized liposome. It only appears
in the time-resolved (TR) spectrum. A 150 µs delay removes
the fluorescence contribution from the antenna and the liposomes
providing a probe that relies only on the emission wavelengths
at 298 nm, 326 nm (the maximum wavelength of the antenna),
and 395 nm, i.e., a wavelength for the direct excitation of Eu3+
(see Figure 1A). The best signal-to-noise (S/N) ratio away from
protein absorption was clearly obtained via energy transfer from
the antenna. This excitation wavelength (326 nm) was then used
for all further studies.
Absorption and Fluorescence Characteristics of Target
Proteins. Three proteins were randomly selected for our studies,
namely, bovine serum albumin, carbonic anhydrase (bovine
erythrocyte), and γ-globulin (bovine serum). Table 1 relates their
absorption and fluorescence characteristics at neutral pH. As
expected, the three proteins show strong absorption and
fluorescence within 200-320 nm and 350-450 nm, respec-
tively. Their absorptivities decrease with increasing wavelength
and become negligible at the excitation (λexc ) 326 nm) and
the emission (λem ) 615 nm) wavelengths of the proposed
sensor. Their fluorescence intensities show similar wavelength
dependence but, under steady-state conditions, interfere with
the reference signal. Because their fluorescence decays are
of Eu3+
.
Figure 3A depicts the TR excitation-emission matrix of the
polymerized liposomes. Although the strongest excitation occurs
between 275 and 325 nm, a wide excitation range is available
to promote luminescence from the lanthanide ion. This versatility
provides ample opportunity for finding an appropriate excitation
wavelength with no matrix interference. Figure 3B compares
the luminescence emitted by the lanthanide ion upon excitation
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10740 J. AM. CHEM. SOC. VOL. 126, NO. 34, 2004

Polymerized Liposomes Bound to Lanthanide Ions
A R T I C L E S
Figure 2. Steady-state excitation and emission spectra recorded from (A)
10-3 M 5As-EDTA-Eu3+ and (B) 92.3 mg‚L-1polymerized liposome
solutions. Both solutions were prepared in 25 mM HEPES. (A) Spectra
were recorded using 2 nm excitation and emission band-pass. (B) Spectra
were recorded using 7 and 2 nm excitation and emission band-pass,
respectively.
Figure 1. Steady-state excitation and emission spectra recorded from (A)
10-3 M Eu-EDTA and (B) 10-6 M 5As solutions. Both solutions were
prepared in 25 mM HEPES. (A) Spectra were recorded using 10 and 5 nm
excitation and emission band-pass, respectively. A cutoff filter was used at
550 nm to avoid second-order emission. (B) Spectra were recorded using
15 and 2 nm excitation and emission band-pass, respectively.
with the following equation: q ) ALN(τH2O-1 - τD2O-1),38 where
q is the number of water molecules in the first coordination
sphere of Eu3+, ALN is the proportionality constant (1.05) for
Eu3+, and τH2O and τD2O are the luminescence lifetimes of the
liposome in H2O and D2O, respectively.
shorter than the fluorescence decay of the polymerized lipo-
somes, using a 150 µs delay also eliminates protein fluorescence
interference. In fact, under these instrumental parameters, there
is no protein contribution to either primary or secondary inner
filter effects.
Assuming that protein-liposome interaction would replace
water molecules in the inner coordination sphere of Eu3+, we
determined the number of available sites for metal-protein
interaction as the number of lanthanide-coordinated water
molecules. Lifetime experiments were carried out with liposome
solutions prepared in aqueous buffer (25 mM HEPES) or in
aqueous buffer: D2O mixtures containing different volume ratios
of H2O-D2O. All measurements were made at λexc/λem ) 326/
615 nm. The lifetime in water (τH2O ) 223.0 ( 7 µs) was
obtained from the average of six independent measurements
directly taken from the polymerized liposomes in aqueous buffer
(25 mM HEPES). The D2O value (τD2O ) 638.8 µs) was
obtained from extrapolation of the linear plot between the
experimental reciprocal luminescence lifetime (τ-1) and the mole
fraction of water (øH2O) in H2O-D2O mixtures (see Figure 4).
The number of coordinated water molecules was calculated as
3.06, which is in good agreement with the fact that EDTA was
synthesized to coordinate five sites in the first coordination
sphere of the lanthanide ion.
Number of Eu3+ Sites Available for Protein Interaction.
Eu3+ can accommodate up to eight or nine molecules of water
in its first (inner) coordination sphere.38 When solvents contain-
ing OH groups are coordinated to Eu3+, efficient nonradiative
deactivation of the excited 5D0 level takes place via weak
vibronic coupling with the vibrational states of the O-H
oscillators. Because the O-H oscillators act independently, the
overall effect on the rate of radiationless deactivation is
proportional to the number of O-H oscillators in the inner
coordination sphere of Eu3+. If the O-H oscillators are replaced
by the lower frequency O-D oscillators, the vibronic deactiva-
tion pathway becomes much less efficient. As a result, the
luminescence lifetime of the lanthanide ion is enhanced, and
the lifetime of the excited state becomes longer. The differences
in the effects of H2O and D2O upon luminescence lifetimes can
be used to determine the number of water molecules coordinated
to Eu3+. The number of water molecules can be determined
Quantitative Analysis with the Polymerized Liposomes.
Similar to the expected effect on the luminescence lifetime, the
(38) Horrocks, W. D., Jr.; Sudnick, D. R. Acc. Chem. Res. 1981, 14, 384.
9
J. AM. CHEM. SOC. VOL. 126, NO. 34, 2004 10741

A R T I C L E S
Santos et al.
Figure 4. Reciprocal luminescence lifetime (τ-1) in µs-1 as a function of
mole fraction of water (øH2O) in D2O-H2O mixtures in Eu-EDTA (1:1)
solution. A ) 1.535 ( 0.1017 µs-1, and B ) τD2O-1 ) 1.565 × 10-3 µs-1
.
Table 2. Analytical Figures of Merita Obtained with the Liposome
Sensor
c
f
g
LDR
(mg‚L-1
γe
RSD
(%)
LOD
(mg‚L-1
)
2 d
proteinb
)
R
(mg‚L-1
)
albumin
CA
γ-globulin
1.8-27.0
1.7-24.5
0.90-18.0
0.9990
0.9992
0.9991
15 620
16 260
47 360
3
3
2
1.8
1.7
0.9
a Analytical figures of merit were obtained with commercial spectro-
fluorimeter. b Protein solutions were prepared in 25 mM HEPES. CA )
carbonic anhydrase. c LDR ) linear dynamic range of calibration curve
defined from the limit of detection to the upper linear concentration. d R )
variance of calibration curve. e γ ) analytical sensitivity defined as the slope
of the calibration curve divided by the precision of measurements at medium
concentration. f RSD ) relative standard deviations at medium concentra-
tions. g LOD ) limit of detection. Values correspond to the protein
concentration giving a signal y ) yB + 3sB, where yB is the average signal
in the absence of protein and sB is its standard deviation.
Figure 3. (A) Time-resolved excitation-emission matrix and (B) time-
resolved luminescence spectra (500-800 nm) recorded at three excitation
wavelengths from a 92.3 mg‚L-1 polymerized liposome solution prepared
in 25 mM HEPES. All spectra were recorded using 30 and 2 nm excitation
and emission band-pass, respectively. Other acquisition parameters were
150 µs delay and 1000 µs integration time. A cutoff filter was used at 450
nm to avoid second-order emission. (B) Excitation spectrum (250-450 nm)
was recorded monitoring the luminescence intensity at 615 nm.
A convenient way to eliminate batch-to-batch variability was
to work with appropriate amounts of liposome that provided
the same 5As-EDTA-Eu3+ concentration in all analytical
samples. The final concentration was chosen to be 5 × 10-6
M. At this concentration, the S/N was 20 and the relative
standard deviation of sixteen determinations (N ) 16) was 2.6%.
Liposome working solutions were prepared upon appropriate
dilutions with HEPES buffer (neutral pH). The dilution factors
were based on the complex concentration in the original
liposome sample. The original concentration was determined
with the method of standard additions. This approach was the
method of choice to compensate for potential matrix interfer-
ence. Different volumes of a concentrated 5As-EDTA-Eu3+
solution were added to several different sample aliquots of the
same liposome volume. The volumes of the standard additions
were negligible in comparison to the liposome volumes to ensure
that the sample matrix was not significantly changed by dilution
with the added standards. The luminescence signal was moni-
tored in the presence of various protein concentrations, and the
measurements were made in batch (25 mH HEPES, neutral pH).
To account for signal stabilization, measurements were done
after 15 min of protein mixing.
Table 1. Absorption and Fluorescence Characteristics of Target
Proteins
b
c
d
λ
max; a
a
326; a615
λ
exc; λem
(nm)
λmax
proteina
(nm; L‚cm-1‚g-1
)
(L‚cm-1‚g-1
)
IR
e
albumin
carbonic
278; 1.9
279; 2.6
0.25; 0.02
0.76; 0.23
269; 342
282; 340
3.6 × 10-4
0.01
anhydrase
γ-globulin
278; 2.4
0.27; 0.01
270; 334
6.6 × 10-4
a Protein solutions (30 mg/L) were prepared in 25 mM HEPES buffer,
b
pH ) 7.0. λmax ) maximum absorption wavelength; aλmax ) absorptivity
at maximum absorption wavelength. c Protein absorptivities at 326 and 615
nm. d Maximum excitation (λexc) and fluorescence (λem) wavelengths. e IR
) intensity ratio between fluorescence at 326 and 615 nm and at the
maximum excitation and emission wavelengths (λexc; λem).
presence of D2O enhanced the luminescence signal of the
polymerized liposomes. The luminescence enhancement was
directly proportional to øD2O. Predicting a similar effect in the
presence of the target proteins, we evaluated the quantitative
performance of the proposed sensor. Initial studies tested the
batch-to-batch reproducibility of the liposome signal. Signal
variations within 1 order of magnitude were observed from batch
to batch. The lack of reproducibility results from different final
concentrations of 5As-EDTA-Eu3+ in the liposome.
Table 2 summaries the analytical figures of merit obtained
for the three proteins. The luminescence intensities plotted in
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Polymerized Liposomes Bound to Lanthanide Ions
A R T I C L E S
Table 3. Comparison of Luminescence Lifetimes Measured with
the Liposome Sensor in the Absence and the Presence of
Proteins
c
proteina
lifetimeb (µs)
RSD (%)
-
233.0 ( 7.0
294.0 ( 7.6
301 ( 8.0
3.1
2.6
2.6
2.1
albumin
γ-globulin
carbonic anhydrase
353.3 ( 7.5
a Protein solutions were mixed with a fixed concentration of liposome
(92.3 mg‚L-1) to provide the following final concentrations: 27 mg‚L-1
of albumin, 24.5 mg‚L-1 of carbonic anhydrase, and 18.0 mg‚L-1 of
γ-globulin. All solutions were prepared in 25 mM HEPES. b Liposome
lifetimes are the averages of six measurements taken from six aliquots of
sample solution. All measurements were made at λexc/λem ) 326/615 nm.
c RSD ) relative standard deviations of luminescence lifetimes.
reference lifetime (absence of protein) to the lifetimes in the
presence of the target proteins. For a confidence level of 95%
(R ) 0.05; N1 ) N2 ) 6),39 the reference value was statistically
different to the lifetime in the presence of proteins, demonstrat-
ing that the lifetime of the liposomes is sufficiently sensitive to
probe the presence of a target protein on the bases of lifetime
analysis. The lifetime in the presence of carbonic anhydrase
was statistically different (P ) 0.05, N1 ) N2 ) 6) to the
lifetimes in the presence of the other two proteins. It is possible,
therefore, to use the liposome sensor to identify carbonic
anhydrase against albumin and γ-globulin. On the other end,
albumin and γ-globulin provided statistically equivalent (P )
0.05, N1 ) N2 ) 6) lifetimes. The inability to differentiate
between these two proteins shows the need for an additional
parameter to improve the selectivity of the proposed sensor
toward a target protein.
Figure 5. Fitted luminescence decay curve for polymerized liposome in
the presence of carbonic anhydrase. Experimental parameters for wavelength-
time matrix collection were the following: λexc/λem ) 326/615 nm, time
delay ) 1000 ns, gate width ) 500 000 ns, gate step ) 40 000 ns, number
of accumulations per spectrum ) 100 laser pulses, number of kinetic series
per wavelength time matrix ) 40, and slit-width of spectrograph: 1 mm.
Polymerized liposome and carbonic anhydrase were mixed in 25 mM
HEPES to produce final concentrations of 92.3 mg‚L-1 and 24.5 mg‚L-1
,
respectively.
the calibration graphs are the average of individual measure-
ments taken from three aliquots of the same working solution.
The linear dynamic ranges of the calibration curves are based
on at least five protein concentrations. The correlation coef-
ficients (R) and the slopes of the log-log plots (data not shown)
are close in unity, demonstrating a linear relationship between
protein concentration and signal intensity. The relative standard
deviations, which are based on individual measurements of six
aliquots of the same working solution, demonstrate the excellent
precision of measurements. The lower limit of detection obtained
for γ-globulin is in agreement with the higher sensitivity of the
sensor toward this protein.
Qualitative Potential of Polymerized Liposomes. Because
no spectral shift is observed in the presence of proteins,
extracting qualitative information from the luminescence spec-
trum of the liposome is not possible. However, the replacement
of OH oscillators by the OD variety causes a significant change
to the luminescence lifetime of the liposome (∆τ ) 415.8 (
17.9 µs). Assuming a similar effect upon protein binding, and
knowing that the luminescence lifetime is usually sensitive to
the microenvironment of the luminophor, we investigated the
feasibility of using this parameter for qualitative analysis of
proteins. The experiments were carried out in batch (25 mM
HEPES) with a fixed concentration of liposome (92.3 mg‚L-1).
The exponential decays were collected at λexc/λem ) 326/615
nm after 15 min of protein mixing. Protein concentrations in
the final mixtures were at the upper limit concentration of their
respective linear dynamic ranges (see Table 2).
Discussion
Our studies demonstrate the feasibility of using the lumines-
cence response of 5As-EDTA-Eu3+ incorporated into polym-
erized liposomes to monitor protein concentrations in aqueous
media. The energy transfer needed for the sensitization of the
lanthanide ion is obtained from the antenna, providing a
reproducible reference signal for protein determination at the
parts per million level. Quantitative analysis is based on the
linear relationship between the luminescence signal of the
liposome and protein concentration. The luminescence enhance-
ment is attributed to the removal of water molecules from the
coordination sphere of Eu3+ upon protein interaction. Although
a straightforward comparison with the limits of detection
reported in the literature is difficult as different instrumental
setups, experimental, and mathematical approaches have been
used for their determination,40-46 our results provide an overall
improvement of at least 1 order of magnitude. Qualitative
analysis is based on the luminescence lifetime of the liposome.
This parameter follows well-behaved single exponential decays
in the absence and the presence of proteins. The shortest lifetime
Figure 5 shows a typical decay in the presence of carbonic
anhydrase. The agreement between the calculated and observed
points over the first two lifetimes of the decays agreed to within
about 1%, and the residuals showed no systematic trends. Single
exponential decays with excellent fittings were also observed
for albumin and γ-globulin. These facts suggest that only one
type of microenvironment per protein surrounds the lanthanide
ion or that only one type of microenvironment significantly
contributes to the observed lifetime. Table 3 compares the
(39) Miller, J. C.; Miller, J. N. Statistics for Analytical Chemistry; Wiley: New
York, 1984.
(40) Malony, K. M.; Shnek, D. R.; Sasaki, D. Y.; Arnold, F. H. Chem. Biol.
1996, 3, 185.
(41) Jelinek, R.; Okada, S.; Norvez, S.; Charych, D. Chem. Biol. 1998, 5, 619.
(42) Hainik, T.; Snejdarkova, M.; Meszar, E.; Krivanek, R.; Tvarozek, V.;
Novotny, I.; Wang, J. Sens. Actuators, B 1999, 57, 201.
(43) Okada, S. Y.; Jelinek, R.; Charych, D. Angew. Chem., Int. Ed. 1999, 38,
655.
(44) Stora, T.; Lakey, J. H.; Vogel, H. Angew. Chem., Int. Ed. 1999, 38, 389.
(45) Cheng, Q.; Peng, T.; Stevens, R. C. J. Am. Chem. Soc. 1999, 121, 6767.
(46) Patolsky, F.; Lichtenstein, A.; Willner, I. J. Am. Chem. Soc. 2000, 122,
418.
9
J. AM. CHEM. SOC. VOL. 126, NO. 34, 2004 10743

A R T I C L E S
Santos et al.
EuCl3‚6H2O (30 mg, 0.082 mmol) was added. It was refluxed for 4 h
and solvent was removed in vacuo to afford the complex as a white
solid (54 mg, 96%). Anal. Calcd for C17H18N3O10‚4H2O.2HCl: C,
28.12; H, 3.86; N, 5.79. Found: C, 27.84; H, 3.64; N, 5.75.
is observed in the absence of proteins, which agrees with our
assumption for luminescence enhancement and protein-lipo-
some interaction. Because the lifetime of the liposome changes
significantly upon protein interaction, the potential for protein
identification on the bases of lifetime analysis exists. However,
the fact that two of the target proteins showed statistically
equivalent lifetimes demonstrates the need for additional
selectivity. Future studies will attempt a selectivity enhancement
based on template polymerization. We will create polymerized
liposomes with templates complementary to the patterns of target
proteins to directly determine them in complex matrixes without
previous separation.
Lipid 1. Polymerizable amine-HCl salt 52 (0.3 g, 0.296 mmol) was
dissolved in dry CHCl3 in the presence of Et3N, followed by the addition
of BOP reagent (0.14 g, 0.32 mmol) and compound 4 (0.17 g, 0.33
mmol). The coupling was carried out at room temperature for 10 h.
The reaction was quenched with saturated NaCl solution. The organic
solvent was removed in vacuo when the product precipitated as a white
solid. It was filtered and washed with water. The pure product was
obtained by silica gel column chromatography with 8% MeOH in CHCl3
1
(Rf ) 0.4) to afford polymerizable ester. Yield: 0.31 g (71%). HNR
(300 MHz, CDCl3-CD3OD): δ 0.91 (t, 6H, J ) 7.1 Hz), 1.25-1.42
(m, 53H), 1.52-1.65 (m, 16H), 2.18-2.29 (m, 16H), 3.40-3.47 (m,
8H), 3.52-3.60 (m, 8H), 3.64-3.66 (m, 4H), 3.71 (s, 6H), 4.13-4.25
(m, 6H), 4.48 (bs, 2H), 4.51-4.56 (m, 1H), 6.94 (d, 1H, J ) 8.5 Hz),
7.32-7.36 (bs, 1H), 7.56 (dd, 1H, J ) 3 and 8.5 Hz), 7.65-7.78 (bs,
1H), 7.77-7.81 (bs, 1H), 8.12 (d, 1H, J ) 8.5 Hz). 13C (125 MHz,
CDCl3-CD3OD): δ 14.13, 14.18, 19.22, 22.74, 25.56, 25.63, 25.71,
25.76, 28.42, 28.88, 28.91, 29.02, 29.05, 29.15, 29.28, 29.39, 29.53,
29.65, 29.68, 31.96, 36.39, 39.21, 39.28, 39.32, 39.42, 41.44, 41.62,
52.24, 53.07, 54.03, 54.24, 55.16, 56.30, 59.08, 60.89, 61.08, 65.27,
65.38, 68.23, 69.55, 69.67, 70.23, 70.28, 70.34, 77.42, 68.23, 69.55,
69.67, 70.23, 70.28, 70.34, 77.42, 77.65, 114.69, 118.02, 118.22, 126.17,
129.37, 170.34, 170.42, 171.37, 171.64, 174.74, 174.82, 175.51, 175.53.
The above ester (0.145 g, 0.99 mmol) was dissolved in CH2Cl2/
THF/MeOH (2/2/4 mL), and solid LiOH (25 mg, 0.99 mmol) was
added. The reaction mixture was stirred at room temperature for 14 h.
The pH of the solution was adjusted to 3.0 with 1 N HCl. The organic
solvents were removed in vacuo, and water was added. The white
precipitated solid was filtered, washed with water, and dried in vacuo.
Yield: 132 mg (92%).1H NMR (500 MHz, CDCl3-CD3OD-D2O):
δ 0.92 (t, 6H, J ) 7.1 Hz), 1.25-1.35 (m, 42H), 1.38-1.45 (m, 6H),
1.53-1.56 (m, 8H), 1.60-1.64 (m, 4H), 2.19-2.23 (m, 4H), 2.26-
2.29 (m, 12H), 3.37-3.44 (, 4H), 3.48-3.54 (m, 10H), 3.57-3.61 (m,
2H), 3.64-3.68 (m, 6H), 3.71-3.74 (m, 4H), 4.52-4.56 (m, 1H), 6.93
(d, 1H, J ) 8.6 Hz), 7.71 (m, 1H), 7.90 (d, 1H, J ) 8.6 Hz).
Lipid.Eu3+ Complex. Lipid 1 (100 mg, 0.072 mmol) was dissolved
in CH3Cl3/MeOH (3/3 mL), and solid EuCl3‚6H2O (26.4 mg, 0.072
mmol) was added. The reaction mixture was stirred for 24 h at room
temperature. The solvents were removed in vacuo, and the white solid
was dried. Yield: 110 mg (100%). Anal. Calcd for C76H118N3O14‚
2H2O: C, 59.15; H, 8.04; N, 6.35. Found: C, 59.47; H, 8.44; N, 6.10.
Instrumentation. Excitation and emission spectra were collected
with a commercial spectrofluorimeter (Photon Technology Interna-
tional). For steady-state measurements, the excitation source was a
continuous-wave 75 W xenon lamp with broadband illumination from
200 to 2000 nm. Detection was made with a photomultiplier tube (PMT,
model 1527) with wavelength range from 185 to 650 nm. The method
of detection was analogue for high signal levels or photon counting
for low signal levels. In analogue mode, the inherent peak-to-peak noise
was 50 × 10-12 A with 0.05 ms time constant. In photon counting
mode, the maximum count rate was 4 MHz, pulse pair resolution 250
ns, rise time 20 ns, and fall time 100 ns with a 220 ns pulse width. For
time-resolved measurements, the excitation source was a pulsed 75 W
xenon lamp (wavelength range from 200 to 2000 nm), variable
repetition rate from 0 to 100 pulses/s, and a pulse width of ap-
proximately 3 µs. Detection was by means of a gated analogue PMT
(model R928) with extended wavelength range from 185 to 900 nm.
SS and TR spectra were collected with excitation and emission
monochromators having the same reciprocal linear dispersion (4
nm‚mm-1) and accuracy ((1 nm with 0.25 nm resolution). Their 1200
grooves/mm gratings were blazed at 300 and 400 nm, respectively.
The instrument was computer controlled using commercial software
(Felix32) specifically designed for the system.
Materials and Methods
Reagents. All reagents and solvents were purchased from com-
mercial suppliers and used without further purification. Nanopure water
was used throughout. Polymerizable 8,10-heneicosadiynoic acid was
used as obtained from GFS Chemicals. Spectroscopy grade organic
solvents from Fisher Scientific were used. All aqueous solutions were
prepared from Nanopure water (Millipore). Experiments were conducted
under an atmosphere of dry nitrogen. For workup, the organic layer
was dried on anhydrous Na2SO4 and concentrated in vacuo. Elemental
analyses were performed by an in-house materials characterization
laboratory. TLC was performed with Absorsil Plus 1P, 20 × 20 cm2
plate, 0.25 µm (Alltech Associates, Inc.). Chromatography plates were
visualized either with UV light or in an iodine chamber. 1H, 13C NMR
spectra were recorded using 300 and 500 MHz spectrometers in one
of the following solvents: CDCl3, D2O, and CD3OD with TMS as
internal standard. 13C NMR spectra data were reported with two digitals
after the decimal to distinguish between close resonance. 13C NMR
spectra of lipid 1 could be studied because of the poor solubility in
common organic solvents.
Compound 4. EDTA ester (1.10 g, 2.92 mmol) was dissolved in
dry ethyl acetate (30 mL), followed by the addition of NHS (0.37 g,
3.22 mmol) and DCC (0.665 g, 3.22 mmol) at room temperature.
Stirring was continued at room temperature for another 12 h. The white
precipitate was filtered under nitrogen and washed with ice-cold ethyl
acetate. Solvent was removed from the filtrate in vacuo. The viscous
liquid was again dissolved in dry CHCl3 (25 mL). 5-Amino salicylic
acid.HCl (0.470 g, 3.07 mmol) was dissolved in DMF (5 mL) in the
presence of Et3N (0.7 mL, 5.03 mmol) and added dropwise. The reaction
mixture was stirred at room temperature for 10 h and quenched with
water. The organic layer was washed with water and dried over Na2-
SO4. The crude product was purified by silica gel column chromatog-
raphy with 15% MeOH in CHCl3 (Rf ) 0.2). Yield: 0.67 g (82%,
1
with respect to consumed EDTA-ester: 0.6 g). H NMR (500 MHz,
CDCl3): δ 1.39 (t, 9H, J ) 7.2 Hz), 2.78-2.95 (m, 4H), 3.49 (s, 2H),
3.58 (s, 6H), 4.20 (q, 4H, J ) 7.2 Hz), 4.25 (q, 2H, J ) 7.2 Hz),
4.34-4.38 (bs, 1H), 6.89 (d, 1H, J ) 8.6 Hz), 8.64 (dd, 1H, J ) 2.7
and 8.6 Hz), 8.12 (d, 1H, J ) 2.7 Hz).
Compound 4 (0.14 g, 0.27 mmol) was dissolved in CH2Cl2/THF/
MeOH (2/4/2 mL), and solid LiOH (85 mg, 2.02 mmol) was added.
The reaction mixture was stirred at room temperature for 15 h. The
pH of the solution was adjusted to 3.0 with 6 N HCl. The solvents
were removed in vacuo, the residue was again dissolved in minimum-
volume MeOH (300 µL), and THF/CH2Cl2 (2/2 mL) was added. The
white precipitate was filtered off and washed with THF and then dried.
1
Yield: 95 mg (69%). H NMR (300 MHz, D2O): δ 3.29-3.34 (m,
2H), 3.47-3.53 (m, 2H), 3.71 (s, 2H), 3.79 (s, 2H), 3.90 (s, 4H), 6.95
(d, 1H, J ) 8.8 Hz), 7.51 (dd, 1H, J ) 3.0 and 8.8 Hz), 7.86 (d, 1H,
J ) 3.0 Hz). Calcd for C17H21N3O10.HCl: C, 43.98; H, 4.74; N, 9.05.
Found: C, 43.71; H, 4.53; N, 8.76.
5As-EDTA-Eu3+ Complex. The acid from the previous step (41
mg, 0.082 mmol) was dissolved in 5 mL of Nanopure water and solid
9
10744 J. AM. CHEM. SOC. VOL. 126, NO. 34, 2004

Polymerized Liposomes Bound to Lanthanide Ions
A R T I C L E S
Luminescence lifetimes were measured with an instrumental setup
mounted in our lab.47 Samples were excited directing the output of a
Northern Lights tunable dye laser (Dakota Technologies, Inc.) through
a KDP frequency-doubling crystal. The dye laser was operated on LDS
698 (Exiton), and it was pumped with the second harmonic of a 10 Hz
Nd:YAG Q-switched solid-state laser (Big Sky Laser Technologies).
Luminescence was detected with a multichannel detector consisting of
a front-illuminated intensified charge fiber-coupled device (ICCD,
Andor Technology). The minimum gate time (full width at half-
maximum) of the intensifier was 2 ns. The CCD had the following
specifications: active area ) 690 × 256 pixels (26 mm2 pixel size
photocathode), dark current ) 0.002 electrons/pixel/s, and readout noise
) four electrons at 20 kHz. The ICCD was mounted at the exit focal
plane of a spectrograph (SPEX 270M) equipped with a 1200 grooves/
mm grating blazed at 500 nm. The system was used in the external
trigger mode. The gating parameters (gate delay, gate width, and the
gate step) were controlled with a digital delay generator (DG535,
Stanford Research Systems, Inc.) via a GPIB interface. Custom
LabView software (National Instruments) was developed in-house for
complete instrumental control and data collection.
Measurements and Procedures. Measurements with the spectro-
fluorimeter were made with standard quartz cuvettes (1 cm × 1 cm).
A fiber optic probe was used with the laser system. The probe assembly
consisted of one excitation and six collection fibers fed into a 1.25 m
long section of copper tubing that provided mechanical support. All
the fibers were 3 m long and 500 µm core diameter silica-clad silica
with polyimide buffer coating (Polymicro Technologies, Inc.). At the
analysis end, the excitation and emission fibers were arranged in a
conventional six-around-one configuration, bundled with vacuum epoxy
(Torr-Seal, Varian) and fed into a metal sleeve for mechanical support.
The copper tubing was flared, stopping a swage nut tapped to allow
for the threading of a 0.75 mL polypropylene sample vial. At the
instrument end, the excitation fiber was positioned in an ST connection
and aligned with the beam of the tunable dye laser, while the emission
fibers were bundled with vacuum epoxy in a slit configuration, fed
into a metal sleeve, and aligned with the entrance slit of the
spectrometer.
Luminescence lifetimes were determined via a three-step proce-
dure:47 (1) collection of full sample and background wavelength-time
matrixes, (2) subtraction of background decay curve from the lumi-
nescence decay curve at the target wavelengths of the sensor, and (3)
fitting the background-corrected data to single exponential decays. The
decay curve data were collected with a minimum 150 µs interval
between opening of the ICCD gate and the rising edge of the laser
pulse, which was sufficient to avoid the need to consider convolution
of the laser pulse with the analyte signal (laser pulse width ) 5 ns). In
addition, the 150 µs delay completely removed the fluorescence of the
sample matrix from the measurement. Fitted decay curves {y ) y0 +
A1 [-exp(x - x0)t1]} were obtained with Origin software (version 5,
Microcal Software, Inc.) by fixing y0 and x0 at a value of zero.
Acknowledgment. This research was supported by the
National Institute of General Medical Sciences (NIH 1 RO1
GM 63204-01A1 to S.M. and A. C.) and the National Science
Foundation (CHE-0138093 to A. C.).
Note Added after ASAP Posting. A spelling error in the
abstract was corrected on August 6, 2004.
(47) Bystol, A. J.; Campiglia, A. D.; Gillispie, G. D. Anal. Chem. 2001, 73,
5762.
JA048963B
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J. AM. CHEM. SOC. VOL. 126, NO. 34, 2004 10745