Quantum dot-fluorescent protein FRET probes for sensing intracellular pH.
ABSTRACT Intracellular pH (pH(i)) plays a critical role in the physiological and pathophysiological processes of cells, and fluorescence imaging using pH-sensitive indicators provides a powerful tool to assess the pH(i) of intact cells and subcellular compartments. Here we describe a nanoparticle-based ratiometric pH sensor, comprising a bright and photostable semiconductor quantum dot (QD) and pH-sensitive fluorescent proteins (FPs), exhibiting dramatically improved sensitivity and photostability compared to BCECF, the most widely used fluorescent dye for pH imaging. We found that Förster resonance energy transfer between the QD and multiple FPs modulates the FP/QD emission ratio, exhibiting a >12-fold change between pH 6 and 8. The modularity of the probe enables customization to specific biological applications through genetic engineering of the FPs, as illustrated by the altered pH range of the probe through mutagenesis of the fluorescent protein. The QD-FP probes facilitate visualization of the acidification of endosomes in living cells following polyarginine-mediated uptake. These probes have the potential to enjoy a wide range of intracellular pH imaging applications that may not be feasible with fluorescent proteins or organic fluorophores alone.
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ABSTRACT: Particle-based nanosensors offer a tool for determining the pH in the endosomal-lysosomal system of living cells. Measurements providing absolute values of pH have so far been restricted by the limited sensitivity range of nanosensors, calibration challenges and the complexity of image analysis. This protocol describes the design and application of a polyacrylamide-based nanosensor (∼60 nm) that covalently incorporates two pH-sensitive fluorophores, fluorescein (FS) and Oregon Green (OG), to broaden the sensitivity range of the sensor (pH 3.1-7.0), and uses the pH-insensitive fluorophore rhodamine as a reference fluorophore. The nanosensors are spontaneously taken up via endocytosis and directed to the lysosomes where dynamic changes in pH can be measured with live-cell confocal microscopy. The most important focus areas of the protocol are the choice of pH-sensitive fluorophores, the design of calibration buffers, the determination of the effective range and especially the description of how to critically evaluate results. The entire procedure typically takes 2-3 weeks.Nature Protocols 12/2014; 9(12):2841-58. · 7.78 Impact Factor
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ABSTRACT: Particle-based fluorescence sensors for the quantification of specific ions can be made by coupling ion-sensitive fluorophores to carrier particles, or by using intrinsically fluorescent particles whose fluorescence properties depend on the concentration of the ions. Despite the advantages of such particle-based sensors for the quantitative detection of ions, such as the possibility to tune the surface chemistry and thus entry portal of the sensor particles to cells, they have also some associated problems. Problems involve for example crosstalk of the ion-sensitive fluorescence read-out with pH, or spectral overlap of the emission spectra of different fluorescent particles in multiplexing formats. Here the benefits of using particle-based fluorescence sensors, their limitations and strategies to overcome these limitations will be described and exemplified with selected examples.Current Opinion in Pharmacology 09/2014; 18:98–103. · 4.23 Impact Factor
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ABSTRACT: We have developed a new ratiometric pH sensor based on poly(N-phenylmaleimide) (PPMI)-containing block copolymer that emits three different fluorescent colors depending on the pH. The strong solvatochromism and tautomerism of the PPMI derivatives enabled precise pH sensing for almost the entire range of the pH scale. Theoretical calculations have predicted largely dissimilar band gaps for the keto, enol, and enolate tautomers of PPMI owing to low dimensional conjugation effects. The tunable emission wavelength and intensity of our sensors, as well as the reversible color switching with high luminescent contrast, were achieved using rational molecular design of PPMI analogues as an innovative platform for accurate H+ detection. The self-assembly of block copolymers on the nanometer length scale was particularly highlighted as a novel prospective means of regulating fluorescence properties while avoiding the self-quenching phenomenon, and this system can be used as a fast responsive pH sensor in versatile device forms.ACS Applied Materials & Interfaces 12/2014; · 5.90 Impact Factor
DENNIS ET AL.VOL. 6
’ NO. 4
March 24, 2012
C2012 American Chemical Society
Quantum Dot?Fluorescent Protein
Allison M. Dennis,†,§Won Jong Rhee,‡David Sotto,†Steven N. Dublin,†,)
and Gang Bao†,*
of Bioengineering, University of Incheon, Incheon 406-772, Republic of Korea.
Laboratory, Los Alamos, New Mexico 87545.
Present address: QIAGEN Hamburg GmbH, Koenigstrasse 4a, 22767 Hamburg, Germany.
cesses, including cell volume regulation,
vesicle trafficking, cellular metabolism, cell
membrane polarity, cellular signaling, and
cell activation, growth, and proliferation.1,2
abnormal pH values in organelles, and low
intracompartmental pH values can dena-
ture proteins or activate enzymes.3Abnor-
mal pHican also affect human physiology
such as the nervous system and pathophys-
iology including cancer4and Alzheimer's
disease.5Monitoring pH changes inside liv-
cellular functions and gaining a better un-
derstanding of physiological and patholog-
Intracellular pH can be measured with a
variety of techniques, including the use of
netic resonance (NMR), absorbance spectros-
copy.2,6,7Fluorescence spectroscopy using
pH-sensitive indicators provides a powerful
tool to assess the pHiof intact cells and sub-
cellular regions, which has several technical
and temporal resolution.3In particular, ratio-
metric measurements, i.e., ratios obtained
from simultaneous (or near simultaneous)
fluorescence measurements at two (or more)
variations in the local probe concentration,
temperature, and optical path length.8High
spatial resolution of pHiindicators is critically
important, since pHimay vary significantly
between subcellular compartments, includ-
ing the cytosol, mitochondria, endoplasmic
While fluorescent indicators based on
small organic dyes have been used to study
viding new insights into nanoparticle-based
§Present address: Centers for Integrated Nanotechnology, Los Alamos National
ntracellular pH (pHi) plays a critical role
in the function of the cell, and its regula-
tion is essential for most cellular pro-
bleaching of these dyes disallow the track-
ing of cellular processes, and how they relate
to pH, over time. Fluorescent indicators with
higher sensitivity, improved signal-to-noise
ratios, and better photostability could enable
with changes in the environment, cell health,
or cell type. In addition, the ability to track pH
temporally and spatially in a living cell could
be utilized for visualizing the endosomal re-
lease of nanoparticle drug carriers, thus pro-
*Address correspondence to
Received for review October 4, 2011
and accepted March 24, 2012.
of cells, and fluorescence imaging using pH-sensitive indicators provides a powerful tool to
assess the pHiof intact cells and subcellular compartments. Here we describe a nanoparticle-
(QD) and pH-sensitive fluorescent proteins (FPs), exhibiting dramatically improved sensitivity
and photostability compared to BCECF, the most widely used fluorescent dye for pH imaging.
WefoundthatFörster resonanceenergy transferbetweentheQDandmultipleFPsmodulates
the probe enables customization to specific biological applications through genetic engineer-
ingofthe FPs,as illustrated by thealtered pH rangeofthe probethroughmutagenesis ofthe
in living cells following polyarginine-mediated uptake. These probes have the potential to
enjoy a wide range of intracellular pH imaging applications that may not be feasible with
fluorescent proteins or organic fluorophores alone.
KEYWORDS: quantum dot.GFP-like fluorescent protein.FRET.pH sensing.
DENNIS ET AL.VOL. 6
’ NO. 4
targeted drug delivery approaches.9,10This informa-
tion is crucial since endosomal release of drug carriers
is necessary to enhance the efficacy of the drug being
Our nanoparticle-based ratiometric pH sensor com-
prises a bright and photostable semiconductor quan-
tum dot (QD) and pH-sensitive fluorescent proteins
(FPs). The QD donor and pH-sensitive FP acceptors
constitute a unique Förster resonance energy transfer
(FRET) pair wherein the environmental sensitivity of
the acceptor fluorophore modulates the emission in-
tensity of the donor. QDs are particularly useful FRET
donors due totheir exceptional brightness, high quan-
tum yields and photostability, the capacity to bind
multiple acceptor molecules, and their broad excita-
tion spectra and narrow, tunable emission spectra.11,12
FPs are versatile FRET acceptors, as the polypeptide
sequence can be genetically modified to include
structural and functional elements necessary for pro-
tein purification, signal transduction, and probe as-
FRET pairs comprising GFP-like FPs and QDs exhibit
high energy transfer efficiencies and enable ratio-
metric measurements, resulting in heightened sensi-
tivity by eliciting opposing changes in fluorescence
emission at two wavelengths, while maintaining an
internal control at an isosbestic point.13?15
RESULTS AND DISCUSSION
Probe Construction and Titration. We developed and
characterized two QD-FP FRET-based pH sensors con-
sisting of carboxyl-functionalized QDs conjugated to
multiple copies of either mOrange, a bright, mono-
mOrange M163K, a mutant with shifted pKa(the pH at
which the measured property is half its maximum) and
improved photostability.17Both the excitation and
emission spectra of the FPs vary with pH due to the
pH dependence of their molar extinction coefficients
(Supplementary Figures S1, S2, and S3). As a result, the
of energy transfer directly correlate to the pH of the
pKaof the acceptor FP. In contrast to an acceptor whose
quantum yield is environmentally sensitive, the pH-
in a probe where both the donor quenching and the
sensitized acceptor emission are affected by changes
change in the ratio of acceptor and donor emission
intensities, thus improving probe sensitivity. With pKa
values of 6.9 and 7.9, respectively, mOrange and mOr-
ange M163K are appropriate acceptors for sensitive
detection in or near the physiological pH range. FPs
were conjugated to QDs via standard carbodiimide
chemistry,18with absorbance spectroscopy indicating
an average of 15.7 and 16.5 proteins per QD for the
mOrange and mOrange M163K probes, respectively
(Figure 1b). This conjugation method covalently links
primary amines in the proteins to carboxylic acids on
the surface of the QDs, ensuring that the probe assem-
bly is not susceptible to changes in pH. This method,
however, does not give full control of the protein
orientation on the surface of the QD. It is also possible
to have protein aggregation or the attachment of FPs
to other FPs already bound to the surface of a QD,
leading to a variety of donor?acceptor distances, as
discussed below. The presence of FPs on the QD sur-
face as confirmed by the absorption spectra (Figure 1b),
dynamic light scattering (DLS) measurements (Supple-
mentary Figure S4), and the evidence that simply
mixing QDs and FPs without conjugation does not
induce FRET signal (Supplementary Figure S6b) de-
surface, although the valence and orientation of FPs
are unknown. Thus, the average numbers of FPs per
QDs are in fact the maximum average number of
proteins bound to each QD, not an exact estimate of
donor?acceptor ratios of the conjugate.
At alkaline pH values, under QD excitation at
of mOrange at 560 nm. With reduction in pH, the
mOrange emission peak intensity decreases and the
mOrange absorbance reversibly modulate the emis-
sion from the pH-insensitive QD (Figure 1, Supplemen-
tary Figures S5 and S6a). The clear isosbestic point at
540 nm could be used to calibrate differences in
conditions between multiple samples. The ratio of
the acceptor (560 nm) to donor (520 nm) emission
peaks (FA/FD) increased by >12-fold between pH 6 and
8 and ∼20-fold over the range of pH values tested
(5?10), with excellent repeatability (Figure 1e, n = 3).
The sigmoidal fit to the data indicates a pKaof 7.0 for
the QD?mOrange probe. No sensitized emission of
mOrange was detectable below pH 6, and the FRET
efficiency was greater than 0.55 for pH values above 8.
Titration of QD?mOrange M163K probes yielded simi-
(Figure 1f and Supplementary Figure S7).
FRET Analysis. Quantitative FRET analysis demon-
strated that overlap integrals and Förster distances
vary with pH in accordance with the pH-dependent
change in the FP optical properties (Figure 2). The pH-
dependent FRET efficiencies were calculated by com-
QDemission intensityat the most acidic measurement
in a titration. Under acidic conditions, the FPs are
“dead” in that at the emission wavelength of the QDs
they do not exhibit the absorption properties neces-
sary for energy transfer. By using this QD emission
DENNIS ET AL.VOL. 6
’ NO. 4
value, rather than the emission of QDs in the absence
of the FPs, we are isolating the pH-dependent energy
transfer from any external factors, such as differences
in concentration and instrument settings, changes to
the QD during the conjugation procedure, or effects
due to the presence of the protein.
We estimated the average donor?acceptor dis-
tance for this system as described in the Methods
section and found that the donor?acceptor distance
calculated is reasonably constant for both probes, as
demonstrated in Figure 2d. However, the estimated
donor?acceptor distances increased slightly with pH
values, most likely an artifact due to the assumptions
we made in the distance calculations. Specifically, the
isthe maximum numberpossibleafter FPconjugation,
rather than a precise value (as discussed above).
Further, our conjugation method resulted in a variety
of FP positions and orientations relative to the QD
surface, suggesting that the estimated donor?acceptor
distance is an average of a significant range of distances.
Nevertheless, the roughly constant donor?acceptor
distance calculated for mOrange?QD probes sup-
ports the hypothesis that, in our QD?FP pH sensors,
changes in the FP optical properties affect the
FRET efficiency, rather than the donor?acceptor dis-
tance. This is in sharp contrast to distance-based
FRET signal transduction, in which the FRET efficien-
cies increase dramatically as the donor?acceptor
distance is shortened.18,19
Photobleaching. Many common pH-sensitive fluoro-
phores are notorious for their lack of photostability.19
Although mOrange suffers from increased photolabil-
ity compared to other GFP-like fluorescent proteins,16
integration of the FP into the FRET probe improved its
useful lifetime dramatically, since QD excitation with
ultraviolet radiation does not directly excite the FP
chromophore. When excited directly with a fluores-
cence microscope, the mOrange signal diminished
>60% in 15 s and 80% under 60 s of continuous illu-
mination. However, it takes >28 min to reduce the sensi-
tized emission of mOrange by 80% under continuous
Figure 1. QD-FP FRET-based pH sensor. (a) Schematic demonstration of the pH-dependent energy transfer between the
high QD signal; at neutral or basic pH, energy transfer is more efficient, producing an enhanced FRET signal. (b) Absorbance
spectroscopy indicates multiple proteins bound to each QD, as depicted in the inset. (c and d) Titration of QD-FP probes
containing the FP acceptors mOrange and mOrange M163K, respectively, showing increased energy transfer at alkaline pHs
emission to donor emission increases with increasing pH for both probes. Data points are means ( standard deviations for
three independent titrations. (f) The changes in the nanoprobe acceptor to donor ratios are compared to the ratiometric
signal change for the pH-sensitive fluorophore BCECF. One representative titration is shown.
DENNIS ET AL.VOL. 6
’ NO. 4
sensitive fluorophore BCECF decreased by 90% after
just 15 s of continuous illumination (Figure 3a). The
and the QD?mOrange M163K FRET probe likewise exhi-
bited a considerably increased useful lifetime through
the FRET mechanism. Consequently, the QD?FP probes
containing mOrange and mOrange M163K exhibited
rather robust FD/FAvalues under the harsh conditions
improved photostability compared to BCECF enables
a wide range of imaging applications, including the
use of time-lapse imaging for real-time tracking of
Intracellular Imaging. Our QD?FP pH probes clearly
exceed the minimum criteria for effective intracellular
and a greater than 30% change in the acceptor to
donor emission ratio.20Importantly, our probes are
most responsive around physiological pH values, and
the excitation and emission wavelengths of the donor
(QD) and acceptor (FP) correspond to common filter
sets, enabling measurements with existing detection
modalities, such as fluorescence microscopes and flow
changes temporally and spatially, we performed live-
cell fluorescence microscopy with a modified QD?
mOrange probe containing a C-terminal polyarginine
sequence for cellular delivery. The inclusion of this
peptide facilitates the endosomal uptake of QD?FP
constructs.21We incubated cultured HeLa cells with
the nanoprobe for an hour, rinsed away unbound
probes, and imaged over several time points using
Figure 2. FRET analysis. (a and b) Calculated overlap integral, J, and Förster distance, R0, as a function of pH for QD-FP FRET
probes containing mOrange and mOrange M163K, respectively. (c) FRET efficiency for both probes over the relevant pH
range. (d) Donor?acceptor distance versus pH for the probes containing mOrange and mOrange M163K. Data shown in (c)
and (d) are representative of one of three independent titrations.
Figure 3. Photostability. The photostability of the fluorescent proteins and FRET-based pH sensors is compared to that of
BCECF, a pH-sensitive fluorophore, during continuous illumination in a fluorescence microscope. (a) Relative intensity of the
emission. (b) Acceptor/donor emission ratio as a function of time for the QD?mOrange and QD?mOrange M163K probes
comparison. The differences at short time scales are highlighted in the insets of (a) and (b). Shown here are representative
results of one of three independent experiments.
DENNIS ET AL. VOL. 6
’ NO. 4
filter sets that selected for (1) the direct excitation and
emission of the QD, (2) the direct excitation and
emission of mOrange, and (3) the FRET signal, i.e.,
excitation of the QD and emission of mOrange. We
endocytotic vesicles to the early endosome to the late
endosome, the drop in pH should induce changes in
the probe signal, decreasing the mOrange and FRET
signals (Figure 4a). This was indeed observed 2 h after
probe delivery, as indicated by the much reduced
(mOrange emission under QD excitation) (Figure 4b),
consistent with the results shown in Figure 1. Although
of FP, there was an estimated 1.5?2-fold increase in QD
to cell). Note that all the fluorescence images in Figure 4
were taken under exactly the same optical conditions,
images by the microscope automatically. The difference
in contrasts in the top and bottom panels of Figure 4
could be due to photobleaching of autofluorescent
biomolecules present in the 10% fetal bovine serum
(FBS) in the cell media; however the exact reason
atically in subsequent cellular imaging studies.
As a negative control, HeLa cells were treated with
bafilomycin A and nocodazole, which inhibit the ma-
turation of the endosome.22We found that inhibition
of endosomal acidification eliminated changes in the
FRET signal from the pH nanosensor 2 h after probe
delivery (Figure 5a), suggesting that changes seen in
Figure 4b were due to pH changes. To rule out the
possibility that the FP signal changes were due to
proteolytic degradation of the fluorescent protein,
we delivered a polyarginine-tagged QD?FP probe
containing the relatively pH-insensitive FP mCherry
into HeLa cells for imaging (Figure 5b, Supplementary
endocytic pathway. FRET efficiency is high in the neutral pH of the extracellular environment and early endosome. FRET
and recoveryof some QD signal. Any probe that escapes the endosome regains its elevatedFRET efficiency in thepH neutral
cytoplasm.(b) Fluorescence microscopy images immediately after delivery of theprobe and two hours postdelivery. The QD
maturation of the endosome.
DENNIS ET AL.VOL. 6
’ NO. 4
Figure S7). The persistence of the mCherry and FRET
signals from the nanoprobe at the later time point
indicates that the barrel structure of GFP-like FPs does
endure the endosomal environment, consistent with
the literature.23For intracellular pH sensing experi-
ments, which typically require less than two hours of
fluorescence microscopy, the potential cytotoxicity of
the QD?FP probes is not a concern (Supplementary
Although the unique optical properties of QDs lead
to improved FRET-based biosensor designs,11,12to
date only limited success has been demonstrated for
intracellular applications of QD-based biosensors,24
including the approaches utilizing the inherent sensi-
tivity of certain QDs to the intracellular environment
(such as ion concentration or pH)25or an energy
transfer mechanism.26,27The probes reported thus far
are not ratiometric and, therefore, lack an internal
probe concentration or optical path length. Other
limited examples of QD-based pH sensing in solution
using FRET lack sensitivity in the physiological pH
range, thus may not be suitable for intracellular pH
sensing.28,29Other sensor designs that utilize both
nanoparticle platforms and pH-sensitive fluorophores
have demonstrated an impressive pH range30?33and
applicability in the intracellular milieu,33but are either
less sensitive (as determined by examining the fold
not report sensitivity inaway that enablescomparison
to the probe described here.33None of these studies
address the photostability issue of the probes. The
strategy of using multiple fluorophores with comple-
mentary pKa values in tandem to extend the pH
sensor's dynamic range works very wellfordye-loaded
polymeric nanoparticles.33A similar extension of the
dynamic range of the probes described here may be
possible by employing multiple FP acceptors with
A primary advantage of this probe design is its
inherent modularity. The customization of FP proper-
ties through genetic engineering enables the devel-
and optical properties. For example, the useful lifetime
of QD?FP probes could be further improved by using
GFP-like fluorescent proteins with photobleaching
half-lives longer than those of mOrange and mOrange
M163K. Other protein variants maintain their optical
properties up to 20 times longer than mOrange.17
Furthermore, the engineering of the FP sensitivities
could result in a range of analytes that could be
monitored using this nanoprobe approach. FPs with
sensitivities to chloride and copper have already been
identified,34,35and screening methods could be used
to develop FPs for use in other environmental sensors.
Conveniently, the methods to modify these FPs are
readily available in any molecular biology lab and do
not rely on proprietary, expensive, or technically ardu-
biosensors could be developed using long-lived FPs
selected for their environmental sensitivities and ap-
propriately color-matched QD donors.
In summary, we have demonstrated the unique
features of the novel QD?FP probes for FRET-based
sensing of pHi, including high sensitivity and wide
dynamic range, ratiometric measurements for internal
the ability to tailor the probe design for different pH
ranges. These probesarewell suitedtoawiderange of
intracellular pH-dependent imaging applications that
are not feasible with fluorescent proteins or organic
fluorophores alone. For example, one could use QD?
nanocarriers for drug/gene delivery and mOrange
M163K probes for pH mapping of the cytosol. We envi-
sion that, by tailoring the FP to the specific application,
Figure 5. Control studies for cellular imaging. (a) Fluores-
cence microscopy images immediately after delivery of the
bafilomycin A and nocodazole, drugs that arrest the en-
dosomal progression. The QD images (left) demonstrate
consolidation of the probe in the endosomes over time;
images of the direct excitation of mOrange (center) and
FRET emission (right) are similar at both time points,
indicating an unchanged pH due to the drug treatment.
(b) Fluorescence microscopy images immediately after
delivery and two hours postdelivery of an mCherry-based
QD?FP probe that shows less pH sensitivity than the
mOrange-based probes. The persistence of the mCherry
and FRET signals after two hours indicates that the probe,
including the fluorescent protein, survives endosomal ma-
DENNIS ET AL.VOL. 6
’ NO. 4
this type of QD?FP FRET probe could be used for
sensitive and multiplexed monitoring of environmental
analytes such as pH and metal ion concentration in
both the intracellular and extracellular environment.
MATERIALS AND METHODS
FRET Probe Preparation. The QD?FP probes were assembled by
incubating a 1 μM solution of 525 nm emitting Qdot ITK carboxyl
USA) with a 40-fold excess of the appropriate protein and a 1500-
fold excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC; Pierce, Rockford, IL, USA) overnight at 4 ?C with gentle
shaking. Byproducts, unreacted EDC, and excess protein were
removed using a centrifugal filtration device with a 100 kDa
molecular weight cutoff (Microcon Ultracel YM-100, Millipore,
Bedford, MA, USA) at 1000 rcf. Dynamic light scattering measure-
ments indicate that the average hydrodynamic diameter of the
QDs is 14.5 ( 1.5 nm and that for the QD?FP probes is
25.1 ( 2.3 nm (Supplemental Figure S4).
FRET Measurements and Analysis. The spectral characteristics of
the FRET probe were measured over a range of pHs by diluting
15 pmol of the probe in 500 μL of 20 mM phosphate-buffered
saline þ 1% (w/v) bovine serum albumin, pH 10.0, and titrating
with 1 N HCl. Fluorescence emission spectra were measured
with a Horiba Jobin Yvon Fluorolog-3 spectrofluorimeter with
bandwidth, and 5 nm stepsize. Following titration with HCl, a
the pH probe. Controls included titration of unconjugated QDs
and fluorescence spectroscopy of a mixture FPs and QDs
(unconjugated) to ensure the pH stability of the QDs and the
conditions, respectively (Supplemental Figure S3).
The QD emission spectrum and the protein excitation
spectra over the range of pHs were used to calculate the
spectral overlap integral:
wavelength in nanometers.36The overlap integral is used to cal-
culate the Förster distance, R0, i.e., the distance between the donor
R06¼ (8:785 ? 10?5)K2QDJη?4
where κ2isthe dipole orientation factor, assumed to be 2/3, QD
The FRET efficiencies (E) over the range of pH values were
calculated using the equation
E ¼ 1 ?FDA
where FDAis the QD emission at 520 nm of a conjugated probe
at the given pH, and F0DAis the QD emission from that same
probe at the most acidic pH measured, i.e., where the energy
efficiency at the most acidic point measured is inherently
defined as zero. In calculating the average donor?acceptor
number of acceptors per donor, n, as determined using absor-
bance spectroscopy (Supplementary Figure S3), was taken into
account, but the Poisson distribution of the actual number of
constructs containing greater than five acceptors per donor:37
Photobleaching. Samples were prepared for photobleaching
experiments by mixing a 0.5 μM solution of conjugated probe
with four times the volume of water-extracted mineral oil to
create bubbles of probe within the oil. The mixture was sealed
fluorescence microscope (Applied Precision, LLC, Issaquah, WA,
USA). mOrange and mOrange M163K were excited directly
using a TRITC filter set (555/28 excitation and 617/63 emission).
The sensitized emission of the mOranges resulting from FRET
was examined by exciting the sample with a DAPI excitation
filter (360/40) while monitoring the fluorescent protein emis-
sion through the TRITC emission filter, while a combination of
the DAPI excitation and GFP emission filters (525/50) was used
to image the QD signal. The intensity value for each time point
was noted as an average of 361 pixels. The background signal
the rate of photobleaching. The same procedure was followed to
measure the photobleaching of 20,70-bis(2-carboxyethyl)-5-(and-
6)-carboxyfluorescein (BCECF; Life Technologies) using the FITC
excitation (490/20) and emission (526/38) filter set.
Intracellular Imaging. HeLa cells were cultured in 8-well Lab-
USA). QD?mOrange?Arg9 or QD?mCherry?Arg9 probes
were delivered by incubation with the cells in Opti-MEM at a
concentration of 50 nM for 1 h at 37 ?C. The cells were then
rinsed three times before being covered with Opti-MEM con-
taining 10% FBS. After delivery, the same cells were monitored
for 2 h with the same optical conditions for each filter set (QD:
DAPI excitation, GFP emission; FP: TRITC excitation and emis-
sion; FRET: DAPI excitation, TRITC emission). The cells were
maintained in a controlled environment at 37 ?C and 5% CO2
throughout imaging. To block the endocytic pathway, cells
were preincubated with 400 nM bafilomycin A and 20 μM
nocodazole in Opti-MEM for 30 min before delivering the
QD?mOrange probes. QD?mOrange?Arg9 probes were then
added to the medium at a final concentration of 50 nM for
Live-cell fluorescence imaging was performed using a
pus 60?, Plan Apo N lens, numerical aperture 1.42, and a
CoolSNAP_HQ2/ICX285 camera. Images were collected at 0.2 μm
Conflict of Interest: The authors declare no competing
Acknowledgment. This work was supported bythe National
Institutes of Health as an NHLBI Program of Excellence in Nano-
Nanomedicine Development Center Award (PN2EY018244 to
GB) and by the National Science Foundation as a Science and
Technology Center Grant (CBET- 0939511).
Supporting Information Available: Details of the protein
preparation and characterization; probe titration; and BCECF
and mCherry characterization. This material is available free of
charge via the Internet at http://pubs.acs.org.
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