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Quantum Dots as Efficient Energy Acceptors in a Time-Resolved Fluoroimmunoassay.

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Communications
Bioanalysis
DOI: 10.1002/anie.200501552
Quantum Dots as Efficient Energy Acceptors in a
Time-Resolved Fluoroimmunoassay**
Niko Hildebrandt,* Loc J. Charbonnire,*
Michael Beck, Raymond F. Ziessel, and
Hans-Gerd L"hmannsr"ben
Dedicated to Professor Herbert Dreeskamp
on the occasion of his 76th birthday
The last decade has witnessed the emergence of semiconductor nanoparticles as very attractive building blocks for
nanotechnology. Spherically or ellipsoidally shaped nanoparticles, also called quantum dots (qdots), display sizedependent optical properties with extremely large absorption
cross sections.[1] When appropriately protected from the
surrounding media by a covering shell, they display narrow
and symmetric emission bands with quantum yields approaching unity in some cases.[2] The inorganic cores of qdots are also
more robust than organic dyes or luminescent proteins toward
photobleaching.[3, 4] Recent accounts have reported on the
possibility of derivatizing qdot surfaces,[5] thereby offering a
broad scope of opportunities for chemical and biological
interactions.[6] These combined features make qdots excellent
tools in analytical techniques associated with fluorescence
spectroscopy, reaching sensitivity limits going down to the
single particle.[4] Qdots display extremely high extinction
coefficients over a wide range of wavelengths as a result of the
absorption of photons with energies higher than the band gap
of the semiconductor material. The resulting excitons (electron–hole pairs) recombine to generate photons with narrow
[*] Dipl.-Ing. (FH) N. Hildebrandt, Dr. M. Beck,
Prof. Dr. H.-G. L)hmannsr)ben
Physikalische Chemie
Institut f0r Chemie und Interdisziplin2res Zentrum f0r Photonik
Universit2t Potsdam
Karl-Liebknecht-Strasse 24–25, 14476 Potsdam–Golm (Germany)
Fax: (+ 49) 331-977-5058
E-mail: hildebr@uni-potsdam.de
Dr. L. J. CharbonniCre, Prof. Dr. R. F. Ziessel
Laboratoire de Chimie MolEculaire
UMR 7509-ECPM-ULP
25, rue Becquerel, 67087 Strasbourg Cedex (France)
Fax: (+ 33) 3-9024-2689
E-mail: charbonn@chimie.u-strasbg.fr
[**] This work was supported by the German Bundesministerium f0r
Wirschaft und Arbeit (InnoNet program 16N0225) and the French
Centre National de la Recherche Scientifique. The authors thank
Frank Sellrie, Universit2t Potsdam, for purification of labeled
streptavidin, Dr. Sophie Haebel, Universit2t Potsdam, for MALDITOF experiments, and Dr. Jennifer Weiss-Wytko and Prof. Jack
Harrowfield, UniversitE Louis Pasteur de Strasbourg, for fruitful
discussions.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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emission bands. These properties make qdots excellent
energy donors in fluorescence (or F.rster) resonance energy
transfer (FRET) experiments,[7] a technique that is ideally
suited for events occurring at the nanometer scale, such as
numerous biological processes. For instance, a homogeneous
maltose sensor has been described in which the maltose
concentration can be monitored by the recovery of qdot
luminescence concomitant to the displacement of a quencher
dye.[8]
Importantly, if qdots are to be used as energy acceptors,
significant direct excitation may be undesirable as it limits the
extent of resonance transfer and results in spurious fluorescence emission. The use of qdots as energy acceptors has
proven possible in a few solid-state devices based on semiconductor quantum wells,[9] with smaller-sized qdots,[10] and
with a blue-emitting polymer[11] as the energy donor, but very
rarely in discrete molecular systems in solution.[12, 13] Mattoussi and co-workers explained low energy-transfer efficiency from organic or inorganic donors to the qdot acceptors
by the fact that the rate of FRET is much slower than the fast
radiative decay rates of the donors.[12]
In the present work, we report that CdSe/ZnS core shell
qdots are excellent energy acceptors in a time-resolved
fluoroimmunoassay system in which a terbium chelate with
long-lived luminescence lifetime is the energy donor and the
strong biotin–streptavidin interaction is used for the recognition process.
The TbL chelate consisted of a terbium cation embedded
in a ligand made of two 6-carboxybipyridyl arms organized on
a glutamate framework (Figure 1). The terminal carboxylate
function of the glutamic residue was activated with the
sodium salt of N-hydroxysulfosuccinimide in the presence of
EDC·HCl (EDC = N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride) according to an existing procedure.[14] The activated ester, TbL*, was incubated with
streptavidin in PBS buffer (pH 7.4) overnight at room
temperature, and the unreacted complex was removed by
extensive dialysis of the protein. The absorption spectrum of
TbL-Strep is a quasi-perfect linear combination of the spectra
of streptavidin and 3.5 TbL complexes (Figure 2). Material
with this labeling ratio was used in all further experiments
(schematically simplified by four TbL labels on streptavidin in
Figure 1).
The MALDI-TOF mass spectra of the labeled tetrameric
streptavidin protein show that the labeling is not simply
localized on one site of each monomeric unit (see Supporting
Information). While a single marker per monomer is the
major species observed, monomers containing zero, two, and
three labels (trace amount) are also evident in the spectrum,
pointing to at least two different environments for the Tb
complexes. Upon excitation in the p!p* transitions centered
on the bipyridine moities at 308 nm, the emission spectrum of
TbL-Strep displays the typical emission lines of the Tb3+
5
D4 !7FJ (J = 0–6) electronic transitions (Figure 3). Excitation
of the ligand resulted in an efficient energy transfer to the
lanthanide cation. The labeling process did not greatly
perturb the Tb environment and, in pure water, the luminescence lifetime of TbL-Strep shows a bi-exponential behavior
with a major long-lived component of 1500 10 ms (97 %) and
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7612 –7615
Angewandte
Chemie
containing 2 % bovine serum
albumin (BSA) at pH 8.3,[15]
the luminescence intensity
decay profile of TbL-Strep
shows two exponential components. The main component remains the long-lived
TbL decay with 1500 50 ms
(60 %), and the second, with
a decay time of 450 30 ms
(40 %), results from TbL species quenched by nonspecific
interactions with BSA. In
this buffer, also used in the
experiments, two distinct
donor species are then presFigure 1. Schematic representation of the assembly of the Biot-QD/TbL-Strep complex. TbL-labeled streptaent. The overall quantum
vidin binds to biotin on the polymer surface of the qdot.
efficiency of the system in
this buffer is 14.5 % (31 % for
pure TbL in water[14]).
As the energy-acceptor component of our fluoroimmunoassay system, an ellipsoidally shaped multilayer CdSe semiconductor core coated with a ZnS protective shell was used.
The complete qdot was coated in a polymer–biotin complex,
providing five to seven biotin molecules per qdot (Figure 1),
with an average long axis of 10–12 nm for the biotinylated
qdots (Biot-QD).[16] These nanoparticles display characteristic absorption and emission spectra with an emission
maximum at 655 nm (Figure 3). Their luminescence decay
profile in the borate buffer is deconvoluted with nanosecondorder multiexponential components (see Supporting Information), associated to the dispersion of the qdot in shape and
size.[17]
Following F.rsterFs theory for resonance energy transFigure 2. UV/Vis absorption spectra of streptavidin (g, c = 4.25
M 107 m), TbL* (d, c = 2.27 M 106 m), and TbL-Strep (c,
fer[18] with a refractive index of n = 1.4 for biomolecules in
c = 1.55 M 107 m) and a linear combination of streptavidin and TbL*
aqueous solution,[19] and assuming that the long-lived excited
(*).
state lifetime of the Tb donor allows for a statistical
distribution of the donor–acceptor dipoles (orientation
factor k2 = 2/3), we calculated the F.rster radius to be R0 =
81 G. This value represents the mean donor–acceptor distance at which half of the energy of the donor is transferred to
the acceptor. As a result of the particularly high e(l)
parameters of qdots, this R0 value is large compared to
those of conventional donor–acceptor pairs (with distances
ranging from 21–61 G)[20] or pairs containing qdots as donors
(47–65 G).[7a, c]
To induce the recognition process and to probe the energy
transfer, increasing amounts of Biot-QD were added to a
solution of TbL-Strep (c = 1 H 109 m). Each sample was
excited with a Xe-Cl excimer laser at 308 nm, and the
emission was measured at 545 and 665 nm, which are the
optimum spectral regions for emission from TbL-Strep and
Biot-QD, respectively. Biot-QD emission was measured at
Figure 3. Normalized emission spectrum (intensity I) of TbL-Strep
665 nm instead of 655 nm (maximum) in order to minimize
(c) and emission (·······) and absorption (a) spectra of Biot-QD.
the weak TbL-Strep background luminescence present at the
latter wavelength (Figure 3). Emission was measured in timeresolved mode, using gated acquisition with a time window
a minor, shorter one of 200 30 ms (3 %) (see Supporting
ranging from 250 to 1000 ms after the excitation pulse, which
Information). The longer lifetime is comparable to the value
resulted in two long-lived intensities I665 and I545. In this way
of pure TbL in water (1.48 ms).[14] In a 50 mm borate buffer
Angew. Chem. Int. Ed. 2005, 44, 7612 –7615
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
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Communications
the autofluorescence of the sample and buffer and interference associated with light scattering in the apparatus are
drastically reduced.[21] Figure 4 shows the dependence of the
intensity ratio R collected at two channels (R = I665/I545) as a
function of Biot-QD added at varying incubation times.
[TbL-Strep] (Figure 4 b) as clear evidence for FRET from
TbL to qdots in the streptavidin-biotin complex.
Further evidence of a sensitization of qdot emission by
energy transfer from terbium was obtained by measuring the
decay profiles at 665 nm for various [Biot-QD]/[TbL-Strep]
ratios (Figure 5). In the presence of TbL-Strep, the qdot
Figure 5. Intensity decay profiles at 665 nm (lexc = 308 nm) for various
values of [Biot-QD]/[TbL-Strep] (0, 0.014, 0.034, 0.071 and 0.25, full
lines from bottom to top, [Biot-QD] = 0–2.0 M 1010 m) and pure BiotQD (dotted line, [Biot-QD] = 1.0 M 1010 m).
Figure 4. a) Intensity ratio R as a function of [Biot-QD]/[TbL-Strep]
between values of 0 and 0.5 at varying incubation times (20 min to
4.5 h). b) R after 4.5 h (equilibrium) for [Biot-QD]/[TbL-Strep] = 0–2.
Initial concentration: [TbL-Strep] = 1 M 109 m. In both graphs R was
normalized to unity for [Biot-QD]/[TbL-Strep] = 0.
Addition of Biot-QD to TbL-Strep led to a strongly
increased R for low qdot concentrations ([Biot-QD]/[TbLStrep] < 0.2) which leveled off at higher qdot concentrations.
The phenomenon evolved slowly with time (Figure 4 a),
reaching equilibrium after 4.5 h (Figure 4 b).[22] Then, the
first part of the curve, corresponding to low qdot concentrations ([Biot-QD]/[TbL-Strep] < 0.1), displayed a marked
rise, mainly originating from a large long-lived qdot emission
increase. At [Biot-QD]/[TbL-Strep] > 0.4, the emission ratio
became almost constant. Extrapolation of the linear rising
part at low concentrations led to an intersection with the
plateau region at [Biot-QD]/[TbL-Strep] 0.13–0.16. This
value is in excellent agreement with the number of biotin
fragments per qdots mentioned by the supplier (5–7),
suggesting that all biotin molecules remain accessible at the
surface of the nanoparticle.
In a control experiment, dynamic energy transfer between
TbL and qdots could be ruled out, as the weak linear increase
of R with [Biot-QD]/[pure TbL] resulted from a stronger
decrease of I545 compared to I665 (see Supporting Information). We thus take the strong increase of R with [Biot-QD]/
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emission profile displays two new long-lived components of
560 40 ms and 170 20 ms, originating from energy transfer
from the two Tb donor components. Given that the intrinsic
Biot-QD decay is very prompt,[17] the long-lived components
observed for Biot-QD in the presence of TbL-Strep can safely
be attributed to the luminescence decays of the donors in the
presence of acceptor, tDAi.[20] Calculation of the energy
transfer rate and the average distance r between donor and
acceptor pairs with multiexponential-decay donors gave a
mean distance of r = 72–76 G depending on the amount of
added Biot-QD (see figure and theoretical treatment in the
Supporting Information).[19] These distances reflect the
assumed system configuration depicted in Figure 1 and
resulted in energy-transfer rates of kET1 = (1.1 0.3) H 103 s1
and kET2 = (3.9 0.6) H 103 s1 as well as an efficiency of E =
63 4 %. It is worth pointing out that this treatment is valid
for a spherically symmetric system consisting of point dipoles
and does not take into account the geometric anisotropy of
the ellipsoidal qdot particles and the nonrandom distribution
of Tb acceptors around the nanocrystals.[24]
In short, these results show that the TbL-qdot pair is a
very promising couple for FRET experiments. The use of
long-lived excited state donors provides two advantages. First,
efficient energy transfer to qdots becomes feasible, and the
competition between radiative deactivation of the donor and
energy transfer[12] swings the balance in favor of the latter.
Second, the increased decay time of the qdot acceptor (up to
some hundred ms) permits the use of a time-resolved
acquisition mode that greatly enhances the sensitivity of the
detection procedure. Under our experimental conditions, a
detection limit of 3.3 H 1012 m was estimated. The large R0
value, which results from the very high qdot absorption,
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2005, 44, 7612 –7615
Angewandte
Chemie
expands the scope of applications of energy-transfer experiments to very big proteins without the need to use large
fluorescent proteins as energy donors or acceptors. Further
work along these lines is currently in progress.
Experimental Section
TbL was prepared according to the literature procedure.[14] Synthesis
and characterization of TbL*: [TbL]Na·4 H2O (45 mg, 55 mmol) was
suspended in 10 mL of dimethyl sulfoxide (DMSO). N-hydroxysulfosuccinimide sodium salt hydrate (14 mg, 60 mmol) and EDC·HCl
(12 mg, 63 mmol) were added, and the solution was stirred at room
temperature for 92 h, resulting in the complete dissolution of the
starting complex. THF was added to precipitate the product. Upon
centrifugation and drying under vacuum, [TbL*]·5 H2O (45 mg, 81 %)
was isolated as a yellow powder. Elemental analysis: calcd for
C33H24N6NaO13STb·5 H2O (%): C 38.99, H 3.37, N 8.27; found: C
39.20, H 3.56, N, 8.39; FAB+-MS: m/z: 682.2 ([TbL*C5H3NNaO7S]+,
95 %), 727.2 ([TbL*C4H3NNaO5S+H]+, 55 %); IR (KBr): ñ = 3447
(s), 1735 (w), 1624 (m), 1388 (w), 1231 (w), 1043 (w) cm1.
TbL-Strep labeling was performed by mixing 0.5 mg of TbL*
(dissolved in 2 mL DMSO) with 1 mg of streptavidin in 200 mL PBS
buffer (pH 7.4). After 1 day of incubation on a shaker at room
temperature the solution was diluted in purified water (Millipore) and
dialyzed.
Biotinylated qdots (Qdot 655 biotin conjugate-trial size lot no.
0603-0050) were supplied by Quantum Dot Corp. (Hayward, CA,
USA). FRET measurements were performed on a modified Kryptor
system (Cezanne, NOmes, France) for time-resolved integrated singlephoton counting at two channels (545 nm and 665 nm; filter-based
wavelength separation) with 2-ms integration steps over 8 ms, with an
OPTex XeCl excimer laser (Lambda Physik AG, G.ttingen, Germany) excitation source at 20 Hz repetition rate. Qdot lifetime
measurements were performed on an Andor iStar ICCD setup with
monochromator and spectrograph wavelength separation (LOTOriel, Darmstadt, Germany) with XeCl excimer laser excitation.
Received: May 6, 2005
Revised: August 8, 2005
Published online: October 25, 2005
.
Keywords: FRET (fluorescence resonance energy transfer) ·
immunoassays · luminescence · quantum dots · terbium
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[22] Preliminary studies on Tb excited-state lifetimes have shown
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Nonspecific binding interactions of BSA and streptavidin that
are able to compete with biotin-streptavidin recognition have
previously been reported.[23] In our case, this nonspecific binding
results in slow equilibration of the system. The interaction of
BSA with streptavidin must first be disrupted before the
thermodynamically favorable TbL-Strep/Biot-QD interaction
can take place. Peeling off the BSA from the Tb emitting species
eliminates the associated quenching processes and is probably
the origin of the rising Tb emission for [Biot-QD]/[TbL-Strep]
< 0.25 (see Supporting Information).
[23] J. H. F. Erkens, S. J. Dieleman, R. A. Dressend.rfer, C. J. Strasburger, J. Steroid Biochem. Mol. Biol. 1998, 67, 153 – 161.
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