Induction of pH sensitivity on the fluorescence lifetime of quantum dots by NIR fluorescent dyes

Article abstract of DOI:10.1021/ja300276s

Modulation of the fluorescence lifetime (FLT) of CdTeSe/ZnS quantum dots (QDs) by near-IR (NIR) organic chromophores represents a new strategy for generating reproducible pH-sensing nanomaterials. The hybrid construct transfers the pH sensitivity of photolabile NIR cyanine dyes to highly emissive and long-lifetime pH-insensitive QDs, thereby inducing a reproducible FLT change from 29 ns at pH >7 to 12 ns at pH <5. This approach provides an unparalleled large dynamic FLT range for pH sensing at NIR wavelengths.

Full text of DOI:10.1021/ja300276s

Communication
pubs.acs.org/JACS
Induction of pH Sensitivity on the Fluorescence Lifetime of Quantum
Dots by NIR Fluorescent Dyes
Rui Tang, Hyeran Lee, and Samuel Achilefu*
Department of Radiology, Washington University, St. Louis, Missouri 63110, United States
S
* Supporting Information
phores.14−19 Although the intrinsic spectral insensitivity of
ABSTRACT: Modulation of the fluorescence lifetime
(FLT) of CdTeSe/ZnS quantum dots (QDs) by near-IR
(NIR) organic chromophores represents a new strategy for
generating reproducible pH-sensing nanomaterials. The
hybrid construct transfers the pH sensitivity of photolabile
NIR cyanine dyes to highly emissive and long-lifetime pH-
insensitive QDs, thereby inducing a reproducible FLT
change from 29 ns at pH >7 to 12 ns at pH <5. This
approach provides an unparalleled large dynamic FLT
range for pH sensing at NIR wavelengths.
QDs to environmental changes favors reproducibility of the
fluorescence data, surface coatings or labeling have been shown
to alter their spectral properties.20 This feature has been used to
develop biosensors where the QD fluorescence intensity is
modulated by organic molecules through energy- or electron-
transfer processes.21−27 Most of these studies have been based
on visible-light-emitting QDs and relied on spectral shifts or
changes in luminescence intensity. Despite the enormous
progress made with these hybrid nanomaterials, the need for
NIR pH sensors with large dynamic measurement parameters
for in vitro, cellular, and in vivo applications remains unmet.
Our goal in this study was to develop a general strategy for
generating photostable NIR pH sensors with large dynamic
pH-sensing capability. The hybrid constructs incorporate the
excellent photophysical properties of QDs with the high pH
sensitivity of NIR organic dyes. Inversion of the pH response
luorescent molecular probes are useful for sensing many
F
chemical and biological processes, such as pH, ligand
binding, enzyme activity, and salt concentrations.1−3 Because of
the critical roles played by the acidity of the medium in
biological systems,4,5 a variety of intracellular pH reporting
strategies with organic dyes have been developed.6 Recent
efforts to translate findings in cell assays to living organisms
have led to concerted efforts to develop pH-sensitive near-IR
(NIR) molecular probes and nanoparticles for assessing the
functional status of both cells and tissue.7−10 These sensors rely
on fluorescence intensity measurements using a reference
calibration model or ratiometric fluorescence techniques. The
inherent difficulties with reproducibility of intensity measure-
ments and the complexity of implementing quantitative in vivo
measurements with emissions at disparate wavelengths
(typically in the visible and NIR regions) have increased the
interest in the use of the fluorescence lifetime (FLT) technique
for pH sensing because the FLT is less dependent on several
factors that perturb intensity measurements.11−13
can be achieved by switching from Forster resonance energy
̈
transfer (FRET) to photoinduced electron transfer (PET)
quenching processes.
We first prepared the pH-sensitive NIR carbocyanine dye
LS662 (Scheme 1) by the method described previously.7 LS662
Scheme 1. Illustration of the Processes Involved in QD pH
Sensing Induced by pH-Sensitive Organic Dyes
Despite these advances, changes in the FLTs of NIR organic
fluorophores in response to pH changes are marginal. Reported
FLT changes between acidic and basic media are <1 ns.
Another major limitation of using NIR organic dyes as pH
sensors for intracellular pH measurements is their poor
photostability, which is amplified in FLT techniques, where a
longer signal integration time is typically needed. An alternative
approach is to use semiconductor quantum dots (QDs), which
offer significant advantages over conventional organic dyes.
These include a broad absorption spectrum for flexibility in
excitation wavelengths; large multiphoton action cross sections
for efficient multiphoton microscopy; narrow, size-tunable
emission for multicolor imaging; superior photostability for
longitudinal studies; high quantum yield for improving
detection sensitivity; and long fluorescence lifetime to minimize
interference from naturally occurring organic fluoro-
exhibits an absorption maximum in the NIR (750 nm) at low
pH that undergoes a hypsochromic shift to the visible region
(520 nm) with increasing pH, with a pKa of 5.2 [Figure S1 in
the Supporting Information (SI)]. Above pH 7, the dye
fluorescence is insignificant. The dye itself is not suitable for
FLT pH measurements because of its <0.2 ns FLT change in
the physiologically useful pH range of 4−7. However, these
Received: January 10, 2012
Published: February 23, 2012
© 2012 American Chemical Society
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Communication
spectral properties are suitable for the development of FRET-
based QD pH sensors through modulation of the emission
properties of the QDs at either 520 or 750 nm with LS662.
CdTeSe QDs coated with ZnS and capped with cysteamine
were synthesized and then conjugated to LS662 (Scheme 1).
The hybrid nanomaterials were purified by either dialysis or
centrifugation followed by removal of the supernatant (see the
SI for detailed synthesis procedures and characterization of
CdTeSe/ZnS QDs and the QD−organic dye conjugates). The
cysteamine ligands served as amine-reactive functionality and
aided the stabilization and solubility of the colloidal QDs in
aqueous media across a wide pH range.
Transmission electron microscopy (TEM) of the QD−dye
conjugates showed an average diameter of 9.4 nm with a fairly
monodisperse distribution (Figure 1A,B). Both the organic
Figure 2. (A) UV−vis absorption spectra of QD750−LS662
conjugates at different pH values. (B) Fluorescence spectra of the
QD750−LS662 conjugates at different pH values. The excitation
wavelength was 488 nm.
from 750 to 520 nm at basic pH produced high QD750
luminescence (Figure 2). The small Stokes shift of the NIR dye,
coupled with the significant overlap of its fluorescence with the
QD750 emission, complicated the determination of the FRET
efficiency using changes in fluorescence intensity. However, this
parameter could be determined using FLT measurements.
Toward this goal, we explored the FLT-based pH sensitivity
of the QD750−dye conjugates in different buffer solutions. The
FLT of the QDs alone has a small pH response, varying
between 29 and 31 ns (Figure S2C). This narrow FLT change
represents <10% of the native QD FLT and does not have the
dynamic FLT range needed for pH sensing in heterogeneous
media. In contrast, the FLT decay profile of the 750 nm
emission of the QD750−dye conjugates showed a significant
pH response (Figure 3A), decreasing from 29 ns at pH >7 to
Figure 1. (A) Representative TEM image of the QD750−LS662
construct. The scale bar is 50 nm. (B) Histogram of the particle sizes
measured from TEM images, giving an average diameter of 9.37
1.13 nm ( 12.1%). (C) DLS data for QD750−LS662, showing a
hydrodynamic diameter of 18.17 2.40 nm ( 13.2%).
surface ligands and the conjugated dye molecules increased the
QD hydrodynamic diameter to ∼18.2 nm (Figure 1C), as
determined by dynamic light scattering (DLS). From
deconvolution of the absorption spectrum of the QD−dye
conjugates, a QD/dye ratio of 1:8 was calculated (Figure S4).
For accurate reporting of the dye-modulated pH response,
the QDs and surface coatings should lack inherent pH
sensitivity and have luminescence near 520 or 750 nm to
match the spectral shift of LS662. Spectral analysis showed that
the luminescence intensity of the parent cysteamine-coated
CdTeSe/ZnS QDs with 750 nm luminescence (QD750) lacked
pH sensitivity (Figure S2). In contrast, the QD750−dye
conjugates displayed a biphasic absorption response to pH
(Figure 2A). Unlike LS662, which exhibited an inverse
relationship with increasing pH at 770 nm, the 750 nm
luminescence of QD750 in the QD750−LS662 conjugates
increased with increasing pH (Figure 2B).
Because the FRET efficiency is determined by the extent of
spectral overlap between the QD emission and the dye
absorption, pH-dependent spectral shifts of the dye absorption
alter the FRET efficiency. On the basis of the spectral
properties of LS662, QD750 luminescence has excellent
spectral overlap with the dye’s NIR absorption at low pH.
This allows energy transfer from the QDs to the dye molecules,
resulting in the quenching of the QD emission. Additional
quenching could be induced by PET from the dye’s tertiary
amine to the QD.28 The expected shift in the dye’s absorption
Figure 3. (A) FLT decay profile of the QD750−LS662 conjugates at
different pH values. (B) Sigmoidal fit of the FLT vs pH curve, giving a
pKa of 5.2.
12 ns at pH <5. A plot of the FLT versus pH showed a
sigmoidal transition with a pKa of 5.2 (Figure 3B). This plot
mirrored that of the dye using fluorescence intensity measure-
ments (Figure S1), exhibiting the same pKa but a reversal of the
intensity versus pH response. This demonstrates that the
organic dye imparted its pH sensitivity to the QDs with high
accuracy, providing a general strategy for improving the
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photostability and signal dynamic range of pH-sensitive organic
molecules.
To determine the FRET efficiency (E) by FLT, we used the
equation E = 1 − τa/τb, where τa is 12 ns (the QD750 FLT at
pH 2, where all of the dye absorption overlaps with the QD750
emission) and τb is 29 ns (determined at pH 10, where no dye
absorption overlaps with the QD750 emission). This gave E ≈
60%, validating the choice of the FLT instead of the emission
intensity as the pH-reporting parameter. At 60%, however,
residual QD emission is still significant, and without a
ratiometric strategy, quantitative pH measurement in cells
and tissue would otherwise be complicated.
A potential strategy for incorporating ratiometric pH sensing
would be to label with LS662 two different QDs emitting at
520 and 750 nm to match the absorption shift of the dye at
different pH. Thus, we prepared QD560−LS662 conjugates,
which were designed to have spectral overlap with the LS662
absorption in neutral to basic pH media (Figure S5A). As
expected, FRET efficiency of this construct increased with
increasing pH, resulting in a reversed FLT trend relative to that
for QD750−LS662 (Figure S5B,C). The 560 nm emission of
the QD560−LS662 conjugates also showed a significant FLT
change from 7 ns at pH >7 to 31 ns at pH <5, with pKa = 5.5
and E ≈ 77% between pH 2 (31 ns) and pH 10 (7 ns). Thus,
FLT alone or a ratiometric pH measurement with the
combination of QD560−LS662 and QD750−LS662 can
successfully be used to determine the pH of chemical and
biological systems quantitatively.
To validate that the induction of the pH sensitivity on the
QDs originated from the pH-sensitive organic dye, we
conjugated QD750 to a non-pH-sensitive dye, LS288 (Figure
S8). This dye has absorption and emission maxima at 760 and
780 nm, respectively. The photophysical properties of this dye,
including the absorption, emission, and FLT of QD750−LS288
construct, were investigated at different pH values (Figure S6).
Upon conjugation of the dye to QD750, a significant pH-
independent emission quenching occurred, resulting in an E
value of ∼73% at all pH values between 2 and 10, where E was
determined by defining τa and τb as the FLTs after (7 ns) and
before (26 ns) dye conjugation. This finding could be used to
develop stable, activatable probes that will restore the QD
fluorescence after removal of the non-pH sensitive-dye by
enzymes or other biological events. More importantly, the
result confirms the role of the organic pH-sensitive dye in
imparting pH sensitivity to the QDs.
To explore the generality of the pH-sensing strategy, we
prepared the pH-sensitive heptamethine dye LS664, which
exhibits fluorescence in an acidic environment that is quenched
by a PET mechanism under neutral and basic conditions
(Scheme 1 and Figure S7). This dye has an absorption
maximum at 760 nm below pH 4 that shifts to 810 nm above
pH 5, generating the nonfluorescent analogue. Both absorption
and emission spectral analyses at different pH values gave a pKa
of 3.2. Using a method similar to the one described for the
synthesis of QD750, we prepared cysteamine-coated CdTeSe/
ZnS QDs emitting at 760 nm (QD760, Figure S9) to overlap
with the absorption maximum of LS664 under acidic
conditions. The QD760−LS664 conjugates were prepared,
and their pH-dependent FLT response was investigated (Figure
4). If FRET were the primary quenching mechanism, we would
expect QD emission quenching at pH <4 and significant
emission intensity at pH >5. Instead, the QD emission
quenching increased with pH, as reflected by the FLT plot
Figure 4. (A) FLT profile of the QD760−LS664 conjugates. The red
solid line is a sigmoidal fit of the data, which gave a pKa of 3.5. (B)
Table of lifetimes obtained from QD760−LS664 conjugates at
different pH; hydrion buffers were used. Two additional points were
added between pH 2 and 4 by titrating a 0.1 M NaCl solution using
HCl or NaOH to optimize the sigmoidal fit of the titration curve.
(Figure 4). The mechanism of luminescence quenching and
enhancement by LS664 is not fully understood at this time, but
previous studies have shown that amines are electron donors to
QDs and could be involved in PET-mediated QD emission
quenching.28 Although QD760 was coated with cysteamine,
which could serve as an electron donor under basic conditions,
we did not observe PET with the analogous QD750 QDs. Since
the major difference between the two QD formulations are the
dyes, we attribute this quenching to PET from the amine
groups on the indolium ring of LS664 to the QDs under basic
conditions. The pKa value obtained from the sigmoidal fit of the
lifetime decay profile was 3.5, which is also consistent with the
pKa of LS664 alone. This demonstrates the feasibility of
applying pH-sensitive dyes with PET capability to modulate the
FLT of QDs. However, delineation of the exact process by
which LS664 perturbs the spectral properties of the QDs needs
further exploration.
In summary, we have developed new pH-sensitive QD−
organic dye hybrid nanomaterials in which the pH-sensing
properties of NIR dyes are imparted to photostable QDs,
resulting in large FLT sensitivity scale. In particular,
perturbation of the FLT of the QDs by FRET or PET
processes illustrates the generality of the approach. The QD
FRET donors can be readily custom-engineered to match the
absorption features of the pH-sensitive dye acceptor used to
modulate the FLT of QDs. This new approach allows
environmentally insensitive QDs to become highly efficient
pH sensors for a variety of potential environmental and
biological applications. For example, the large two-photon (2P)
action cross sections of QDs and the deep tissue penetration of
NIR light ideally position these sensors for use in multiphoton
microscopy of molecular processes across large spatial scales
ranging from cells to thick tissue. Moreover, direct 2P
excitation of the highly photostable QDs prevents the rapid
photobleaching of the pH-sensitive cyanine NIR dyes, which
readily occurs under irradiation with high-intensity lasers.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed experimental methods, dye structures, spectral proper-
ties of different constructs, deconvolution of the spectra, and
pKa measurements. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
achilefus@mir.wustl.edu
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(27) Goldman, E. R.; Medintz, I. L.; Whitley, J. L.; Hayhurst, A.;
Clapp, A. R.; Uyeda, H. T.; Deschamps, J. R.; Lassman, M. E.;
Mattoussi, H. J. Am. Chem. Soc. 2005, 127, 6744.
Notes
The authors declare no competing financial interest.
(28) Tomasulo, M.; Yildiz, I.; Kaanumalle, S. L.; Raymo, F. M.
Langmuir 2006, 22, 10284.
ACKNOWLEDGMENTS
■
This study was based upon work supported by the NIH Grants
R01 EB007276 and R01 EB008111; and in part by R33
CA123537, U54 CA136398 (Network for Translational
Research), and HHSN268201000046C (NHLBI Program of
Excellence in Nano-technology).
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