Cascade Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the Selective Detection of Cysteine

Article abstract of DOI:10.1021/acs.analchem.7b01384

The ability to detect and visualize cellular events and their associated target biological analytes through use of cell-permeable profluorogenic probes is dependent on the availability of activatable probes that respond rapidly and selectively to target analytes by production of fluorescent reporting molecules whose excitation and emission energies span a broad range. Herein is described a new probe, DCM-Cys, that preferentially reacts with cysteine to form a dicyanomethylene-4H-pyran (DCM) reporter whose red-energy fluorescence can be stimulated by two-photon, near-infrared excitation so as to provide visualization of cysteine presence inside living human cells with a high signal-to-background ratio. These aforementioned characteristics and the ability of DCM-Cys to provide selective, nanomolar-level in vitro cysteine detection, as demonstrated by its lack of significant response to other thiols and potential interfering agents from biological environments, are attributed to the molecular designs of the DCM-Cys probe and DCM reporter. Attachment of an acryl moiety to the DCM reporter via a self-eliminating, electron-withdrawing benzyl alcohol-carbamate linker offers a probe having selective, sensitive reaction with cysteine to rapidly produce a reporter whose energies of excitation and emission (λabs report = 480 nm, λemis^report = 640 nm) are red-shifted from those of the DCM-Cys probe (λabs^probe = 440 nm, λ_emis^probe = 550 nm), thereby leading to low background signal from abundant probe and a large signal from the resulting reporter of cysteine presence.

Full text of DOI:10.1021/acs.analchem.7b01384

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Article
Cascade Reaction-Based, Near-Infrared Multiphoton
Fluorescent Probe for the Selective Detection of Cysteine
Rasika R Nawimanage, Bijeta Prasai, Suraj U. Hettiarachchi, and Robin L. McCarley
Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 16 May 2017
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Analytical Chemistry
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Cascade Reaction-Based, Near-Infrared Multiphoton
Fluorescent Probe for the Selective Detection of Cysteine
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Rasika R. Nawimanage, Bijeta Prasai, Suraj U. Hettiarachchi, and Robin L. McCarley*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, United States
ABSTRACT: The ability to detect and visualize cellular events and their associated target biological analytes through use
of cell-permeable pro-fluorogenic probes is dependent on the availability of activatable probes that respond rapidly and
selectively to target analytes by production of fluorescent reporting molecules whose excitation and emission energies
span a broad range. Herein is described a new probe DCM–Cys that preferentially reacts with cysteine to form a dicy-
anomethylene-4H-pyran (DCM) reporter whose red-energy fluorescence can be stimulated by two-photon, near-infrared
excitation so as to provide visualization of cysteine presence inside living human cells with a high signal-to-background
ratio. These aforementioned characteristics and the ability of DCM–Cys to provide selective, nanomolar-level in vitro
cysteine detection, as demonstrated by its lack of significant response to other thiols and potential interferents from bio-
logical environments, are attributed to the molecular designs of the DCM–Cys probe and DCM reporter. Attachment of
an acryl moiety to the DCM reporter via a self-eliminating, electron-withdrawing benzyl alcohol–carbamate linker offers a
probe having selective, sensitive reaction with cysteine to rapidly produce a reporter whose energies of excitation and
report
probe
= 550
emis
emission (
λ
report= 480 nm, λ emis = 640 nm) are red-shifted from those of the DCM–Cys probe (λparobbse = 440 nm,
λ
abs
nm), thereby leading to low background signal from abundant probe, and a large signal from the resulting reporter of
cysteine
presence.
most of these cysteine-selective probes have not had re-
ported their response to the possible cross reactant H2S,8
an important cellular gasotransmitter. And although a
class-selective bio–thiol probe has been shown to be un-
reactive toward a prevalent thiol-containing protein,11 to
date there have been no reports of the potential role of
protein thiols as interferents with cysteine-selective
probes. Finally, there is a great need for probes capable of
selectively detecting cysteine without the requirement of
F
luorescence-based small molecule probes have recently
received much attention for their use in tracking of im-
portant analytes in cellular environments, biological flu-
ids, and in living tissues, without affecting sample integri-
ty. By making use of the intrinsically unique reactivity of
select classes of organic molecules, these probes can be
tailored to detect either broad classes of analytes or spe-
cific targets as a result of analyte-induced formation of
fluorescent reporters.1,2-4
multiple
cysteine
molecules reacting with the probe9 or probe/reporter
excitation and emission energies lying solely outside the
red region.6
The sulfur-containing small molecules glutathione
(GSH), cysteine (Cys), and homocysteine (Hcy) are in-
volved in a number of key biological processes.5 Cys and
Hcy are clearly prominent among the other naturally oc-
curring amino acids, as they are the only ones with a free
thiol (–SH) moiety. Hcy and Cys differ from each other
by the presence of a single methylene unit in their side
chain, giving them similar structural and chemical char-
acteristics. Owing to their near-identical chemical reac-
tivities, the selective detection, quantification, or visuali-
zation of a single thiol analyte by discriminating against
other thiol-containing small molecules remains a great
challenge.
Fluorescent dyes with excitation and emission energies
in the red and NIR range (640–900 nm) possess unique
advantages for tracking molecular processes in vitro and
in vivo with less background interference.12 NIR dyes of-
fer increased penetration distance of light, thereby facili-
tating deep-tissue imaging with minimal damage to bio-
logical components. Although long-wavelength cyanine
dyes are widely used in designing NIR sensors for bio-
imaging, they typically suffer from poor photostability in
aqueous solutions,13 thereby limiting their universal appli-
cation
for
Only a select few of the extant thiol-responsive
probe/reporter systems have the ability to preferentially
detect cysteine versus Hcy and GSH.6-10 Furthermore,
analyte detection, particularly those aimed at real-time
assessments.
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Dicyanomethylene-4H-pyrans and their derivatives two-photon absorption cross section in the NIR
region18 and emits in the red and NIR region, both key
for tissue imaging applications. As a result, DCM–Cys
offers quick (<10 min) and highly selective in vitro de-
tection of cysteine at sub-nanomolar levels, and it pro-
vides for high-fidelity detection of cysteine in living
mammalian cells.
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(DCM dyes) have long been used for non-linear optical
materials, logic gates,14 and photovoltaic sensitizers.15,16
As
a
donor–
π–acceptor type chromophore, derivatives of DCM
have attractive photophysical properties, such as tunable
emission wavelength in the red or NIR region via control
over electron donor ability, large intramolecular charge-
transfer-based Stokes shift, and high photostability.14
ꢀ
EXPERIMENTAL SECTION
9
Materials and General Methods. All chemicals and
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instrumentation were obtained from commercial suppli-
ers. Stock solutions of the DCM–Cys probe were made in
DMSO (Sigma-Aldrich). Water from
NANOpure Diamond water System (18 M
a
Barnstead
ꢀ
cm) was used
to prepare phosphate-buffered saline (PBS) required for
those experiments requiring its use.
The DCM reporter and DCM–Cys probe were synthe-
sized as outlined in the synthetic scheme of the Support-
ing Information. The structures and purities of the probe
1
and the reporter were confirmed with ESI-MS, H NMR,
1
and 13C NMR. The H NMR and 13C NMR spectra were
recorded in chloroform-d at room temperature using
Bruker AV-400 and AV-500 spectrometers.
Spectroscopic Methods. All spectroscopic meas-
urements were performed in 0.1 M, pH 7.4 phosphate-
buffered saline solution, using 1-cm × 1-cm quartz cu-
vettes (1.5-mL volume, Sigma-Aldrich). Fluorescence
data were collected using a Perkin Elmer LS-55 spec-
trometer, and absorption spectra were obtained with a
Varian Cary-50 spectrophotometer.
Figure 1. Proposed cysteineꢀinduced, turnꢀon route of DCM-
Cys probe to yield the corresponding fluorescent DCM reporter.
Procedure for Solution-phase Thiol Sensing. Stock
solutions of 2 × 10−4 M DCM–Cys probe were prepared in
100% DMSO and was subsequently diluted to prepare
preferred concentration solutions in pH 7.4 PBS/DMSO
buffer. Stock solutions of thiol were freshly prepared prior
to each experiment. For the calibration curve of cysteine,
a fixed concentration solution of DCM–Cys was incubat-
Furthermore, compared to traditional dyes that undergo
only single-photon excitation, DCM derivatives have sig-
nificant two-photon absorption cross sections.17-19 As
such, use of DCM-based dyes in two-photon microscopy
studies18 is predicted to allow for deeper penetration of
the lower energy photons into tissues.4
°
ed with different concentrations of cysteine at 37 C for 15
The work here presents the design, synthesis and
evaluation of a fluorescent probe, DCM-Cys, for the
selective detection of cysteine under physiological
conditions and in living cells. The general design ra-
tionale of the cysteine-activatable, DCM–Cys is de-
scribed as follows. The probe contains a reporter dye
attached to a cysteine-reactive acryl moiety20 via a car-
bamate-based, self-eliminating linker that is not
prone11,21 to potential esterase-catalyzed hydrolysis
within cells,22,23 Figure 1. Initially, in the presence of
cysteine, the latter’s sulfhydryl group will undergo Mi-
min, and spectral data were recorded. For the DCM–Cys
probe (detect the release of the free dye), samples were
excited at 520 nm, and emission was detected at 640 nm.
The excitation and emission slit widths were 5 nm and 10
nm respectively.
Cell Culture. Human non-small cell lung cancer cells
H596, cell culture base media, and fetal bovine serum
(FBS) were purchased from American Type Cell Culture
(ATCC, Manassas, VA). All cell culture procedures were
performed as suggested by ATCC. H596 cells were cul-
tured in RPMI-1640 with 10% FBS and 10 IU mL−1 penicil-
°
chael addition to the
α,β-unsaturated double bond in
lin-streptomycin. Cells were incubated at 37 C in a hu-
the reactive acryl moiety to form a thio-ether. The se-
cond step utilizes the amine group of the cysteine ami-
no acid, which makes a nucleophilic attack on the car-
bonyl carbon of the linker, thereby forming a cyclized
thio-lactam product. Lactam formation will induce a
cascade of downstream processes involving quinone
methide formation that results in release of the linker,
carbon dioxide, and highly fluorescent DCM reporter,
Figure 1 and Scheme S4. The DCM reporter has a high
midified incubator containing 5% wt/vol CO2.
Imaging of Cysteine in Cells via Scanning Laser
Confocal Microscopy. H596 cells were cultured over-
night on 22-mm × 22-mm glass cover slips on a treated
tissue culture 6-well plate purchased from Fisher Scien-
tific. The existing growth medium was replaced with 2 mL
of fresh cysteine free medium and then incubated at 37 °C.
Solutions of DCM–Cys dissolved in DMSO were added to
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Analytical Chemistry
the cells to give a final DCM–Cys concentration of 3 × 10−5
1
2
M, with the DMSO concentration at < 1%. Cells were in-
°
cubated with DCM–Cys at 37 C for 10 min. For confocal
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microscopy of fixed cells, the medium was removed, and
the cells were fixed in 2 mL of 4% paraformaldehyde for 15
min with shaking. After fixing, the cells were rinsed with
Nanopure water, and the cover slips were mounted to
glass slides with Immumount (obtained from Fisher Sci-
entific). Glass slides were left in the dark overnight to
allow the Immumount to dry.
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Confocal images were acquired using a Leica TCS SP2
spectral confocal microscope. For images of cells exposed
to DCM-Cys, samples were excited using the 488-nm line
of an Ar/KrAr laser (laser intensity = 45.32%), and the
spectral emission was collected between 625 and 750 nm
(Leica DD488/543); PMT voltage = 710 V. All images were
collected using a pinhole of 1.0 Airy units. In the control
experiment, cellular thiols were scavenged by pre-
°
incubating with 10 mM N-ethylmaleimide (NEM) at 37 C
for 30 min. The cells were then washed with 0.1 M, pH 7.4
PBS to remove any excess NEM, followed by incubation
with 3 × 10−5 M DCM–Cys prior to being fixed, as de-
scribed above.
For live cell multiphoton microscope work, cells were
imaged as outlined below. In those cases where NEM was
used, the NEM remained in the solution bathing the cells
during probe exposure. Solutions of DCM–Cys dissolved
in DMSO were added to the cells to give a final DCM–Cys
concentration of 2 × 10−5 M, with the DMSO concentra-
tion at < 1%. Cells were incubated with DCM–Cys at 37 °C
for 10 min. All the multiphoton images were obtained
using a Leica TCS SP5 spectral multiphoton microscope.
For images of cells exposed to DCM-Cys, samples were
excited using a MaiTai laser tuned to 900 nm (2.11 W laser
power, mode-locked Ti: sapphire), and emission was col-
lected between 600–700 nm at a PMT gain of 250 V (Ob-
jective used: 63.0 × 1.20, Water UV).
Figure 2.
Absorption and emission spectra of 30 ꢁM
DCM–Cys probe and 10 ꢁM DCM reporter in 40% DMSO:
60% 0.1 M, pH 7.4 phosphateꢀbuffered saline at T = 37 °C.
tion band in comparison to that of the DCM–Cys probe,
with the maximum absorbance located at
εre4p8o0rt= 27,500 M−1 cm−1). In addition, the reporter pos-
sesses a large Stokes-shifted emission, with a peak wave-
λ
report= 480 nm
abs
(
report
length (
λ
) at 640 nm.
emis
Images were frame and line averaged 4 times with a
zoom factor 0f 3.25. Image analysis was performed using
Fiji, ImageJ, and Leica LAS AF lite software. Statistical
evaluation of cytosolic intensities from obtained images
was performed using Fiji/ImageJ, where individual cells
were traced and the integrated intensity was measured
within the region-of-interest (ROI).
A key issue our work addresses is the ability to obtain
high reporter signal and low background response from
unreacted probe, so that high signal-to-background
measurements are possible when substantial quantities of
unreacted probe are present. The design of the DCM–
Cys probe leads to it not having a substantial absorptivity
report
=
abs
at the energy of excitation for the DCM reporter (
λ
ꢀ
RESULTS AND DISCUSSION
480 nm), with the absorptivity of the probe at 480 nm
being only 4% that of the reporter. Also, the fluorescence
emission intensity of the probe is substantially lower than
that of the reporter at the emission maximum of the re-
porter. The large difference in probe and reporter proper-
ties results from the electron-withdrawing carbamate
linkage in the DCM–Cys probe, a tack we recently dis-
Spectroscopic Characteristics of the Designed
Probe/Reporter System. In order to determine the spec-
tral properties of the probe/reporter system, absorption
and fluorescence emission spectra of DCM–Cys probe
and free DCM reporter were obtained in aqueous
DMSO/PBS solution. As seen in Figure 2, the DCM–Cys
probe has a weak absorption band with a maximum ab-
closed for an enzyme-activatable naphthalimide probe.21
sorbance (λparobbse) centered near 440 nm (ε 440 = 2300 M−1
probe
report
In total, excitation of the probe at
λ
yields very little
abs
cm−1). When DCM–Cys was excited at its wavelength of
report
background signal at λ emis that is assignable to unreacted
probe presence. Furthermore, the remarkable ~160 nm
difference in the emission and absorption maxima of
DCM is an added advantage, as such a large Stoke’s shift
maximum absorbance, a fluorescence spectrum was ob-
probe
tained with a peak fluorescence (
λ
) centered around
emis
550 nm. The free DCM reporter has a very strong absorp-
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Page 4 of 8
will facilitate the efficient monitoring of reporter release with DCM-Cys, demonstrating the rapid nature of the
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in cells where excitation light scattering events can con-
tribute substantially to background interference.24
known thiolactam cyclization reaction when cysteine re-
acts with the acryl moiety attached to a good leaving
group.20 In total, the observed mass spectral signals sup-
port formation of the DCM reporter via the proposed
mechanism in Scheme S4.
In order to determine the capability of DCM–Cys to act
as a turn-on probe upon its reaction with cysteine, the
fluorescence emission was monitored for an aqueous so-
To determine whether the DCM–Cys fluorescence
turn-on response is a function of cysteine concentration
in the media, varying concentrations of cysteine were
lution of 30 ꢁM DCM–Cys and 60 ꢁM cysteine. As noted
9
incubated with 30 ꢁM DCM–Cys solution for 20 min at 37
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°C, and the turn-on response at 640 nm was measured.
As shown in Figure 4, it is clear that the fluorescence re-
sponse increases with increasing concentration of cyste-
Figure 3. Timeꢀdependent fluorescence (λex = 520 nm, λem
=
640 nm) of 30 ꢁM DCM-Cys incubated with 60 ꢁM cysteine in
40% DMSO: 60% 0.1 M, pH 7.4 phosphateꢀbuffered saline
(PBS) at T = 37 °C; black arrow indicates introduction of cysteꢀ
ine at ~220 s. Inset: a) emission spectra of 30 ꢁM DCM-Cys
after cysteine addition, ꢂt = 2 min; b) visualization of the fluoꢀ
rescence change (λex ≈ 365 nm) before and 10 min after cysteine
was added, T = 37 °C.
Figure 4. Fluorescence response of DCM-Cys probe (30 ꢁM)
upon 20ꢀmin incubation with [cysteine] = 0–30 ꢁM. Inset: plot
of resulting fluorescence intensity (λex = 520 nm/λem = 640 nm)
vs concentration of cysteine; R = 0.98. Values shown in Inset are
the average of 3 trials, with error bars signifying ±1 sample
standard deviation. T = 37 °C.
in Figure 3, a rapid increase in fluorescence was observed
in both the time-dependent fluorescence emission and
the time-lapse wavelength scans from 550 nm to 750 nm.
The observed fluorescence band centered near 640 nm
(Figure 3, inset a) is consistent with that of the free DCM
fluorescence (Figure 2), indicating release of the free re-
porter. Production of fluorescent reporter was readily
visualized, Figure 3, inset b. Maximal cysteine-stimulated
release of DCM reporter occurred within about 700 s,
with a fluorescence turn-on value of 15 being achieved.
ine in the media. From the plot of fluorescence intensity
at 640 nm as a function of concentration, (Figure 4, inset)
it is concluded that there exists a linear relationship (r =
0.97511; slope = 0.889 ± 0.056) between cysteine concen-
tration and fluorescence response of the 30
ꢁM probe
solution, up to 30 M cysteine (1 equivalent). From analy-
ꢁ
sis of this data set, we obtained a concentration limit of
detection of 21 nM and concentration limit of quantifica-
tion of 70 nM.
Mass spectrometry evaluation (ESI-MS, negative- and
positive-ion modes) of DCM–Cys solutions exposed to
°
cysteine at 37 C for 30 min demonstrated formation of
GSH, Hcy and H2S—the other commonly found thiols
in mammalian systems with chemical reactivities similar
to that of cysteine—have the potential to act as cross-
reactive species with the DCM–Cys probe. The pKas of
GSH and Hcy are almost the same (8.72, 8.25, respective-
ly) as that of cysteine (8.10),26 implying similar potential
reactivity during the first addition step to yield their cor-
responding thioethers, Scheme S4. H2S on the other
hand, which is considered more acidic (pKa 6.9) than the
other three, could act as a stronger nucleophile. Also,
proteins with free cysteine residues could act as possible
reactive species with the DCM–Cys probe; the protein
concentration within cells is ~20–25 wt%.27 The most
abundant source of thiols in blood that are not within
the DCM reporter and the thio-lactam. As shown in Fig-
ure S2, free DCM reporter release (Figure 1, Scheme S4)
was noted by the [M−H]− signal at m/z = 310.0978 (ex-
pected m/z = 310.0979, 0.32 ppm error). In addition, pro-
duction of the expected cyclized thio-lactam product was
observed by the [M+H]+ signal at m/z = 176.0371 (expected
m/z = 176.0383, 6.8 ppm error).25 There was no signal
observed for DCM–Cys probe (expected m/z = 516.1561
[M+H]+ or m/z = 514.51700 [M−H]−), pointing to the lack
of any remaining unreacted probe, which is in agreement
with outcomes in Figure 3. Furthermore, we did not find
evidence for presence of the DCM-Cys thioether addition
product (Scheme S4) after 30-min incubation of cysteine
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Analytical Chemistry
achieved with cysteine at 1200 s incubation time, and only
cells is the result of serum albumin presence.28 Similar to
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BSA, human serum albumin (HSA), possesses only one
free cysteine (Cys 34)29 as a potential site of reaction with
DCM-Cys. Furthermore, BSA does not suffer from the
storage-induced fluxional free thiol content of Cys 34 in
2.5% that of cysteine at 300 s incubation; such responses
are not a concern with respect to selective detection of
Cys in mammalian cells, as the intracellular levels of Hcy
are dwarfed by that of Cys, with [Hcy] being roughly 0.5%
that of [Cys].30 Similarly, GSH yields a signal at 300 s and
12oo s that is 1.2% and 4.9% that achieved with cysteine,
and the responses from H2S and BSA are virtually un-
changed after their exposure to the DCM–Cys probe. The
high probe responsivity toward cysteine versus GSH and
Hcy under the conditions of Figure 5 is posited to result
from differences in ring-formation kinetics, Scheme S3,20
with the most rapid ring formation occurring for Cys.
Formation of the smaller 7-membered lactam ring will be
kinetically favored over that of the larger 8- and 12-
membered rings in the cases of Hcy and GSH.20 We pro-
pose the lack of any observable reaction with H2S is due
to its inability to undergo ring formation. The lower reac-
tivity of cysteine residues (Cys-34) in BSA may be due to
steric hindrance of the Cys residue as a result of its being
found nearer the inside of the protein;5 to our knowledge,
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this
is
the
only
cysteine-
selective fluorescence probe whose potential reactivity with
serum albumin has been examined and reported.
Figure 5. Time-dependent fluorescence (λex = 520 nm, λem
=
640 nm) from 30 ꢁM DCM-Cys incubated with 60 ꢁM sul-
fur-based analytes in 0.1 M, pH 7.4 PBS/DMSO (60%:40%)
at
It is important to note that the lack of an appreciable
signal at 640 nm for reporter production upon probe ex-
posure to individual possible interferents in Figure 5 may
not be a fair indicator of probe selectivity toward cysteine.
In particular, the low probe response for GSH, which
makes up over 90% of intracellular non-protein thiols,
results from its slow ring formation kinetics after GSH
interaction with the acryl group to yield the thioether,
Scheme S4. Within mammalian cells, in particular cancer
cells, the concentration of GSH is typically in the 1–3 mM
37 °C. The signal versus that achieved with cysteine alone
HSA.29
As shown in Figure 5, these components (60
incubated with 30 M DCM-Cys, and the fluorescence
signal was examined over a 20-min time period. Cysteine
ꢁM) were
ꢁ
̴
is the fastest reacting species, exhibiting a rapid turn-on
response that reached a maximal value in less than 12
min, a characteristic not exhibited by the other species in
the 20-min evaluation period, the longest time examined.
Thus, none of the potential cross-reactive thiol compo-
nents tested exhibits a significant ability to act as an in-
terferent with respect to fluorescence signal production,
in comparison to the response for cysteine at 640 nm.
Specifically, Hcy yields a response that is 14% of the signal
range and that of Cys is approximately 100 ꢁ
M.31-34 Under
such conditions, it is possible any GSH–probe thioether
formed will result in decreased sensitivity of DCM–Cys
probe response to the ~10-fold lower Cys concentration.
To examine a scenario more representative of that in
cells, we exposed DCM–Cys to a solution composed of
both 1 mM GSH and 100
tion containing only 1 mM GSH was also obtained. Upon
plotting the time-
ꢁM Cys; the response to a solu-
dependent fluorescence response at 640 nm (λex = 520
nm) resulting from probe incubation with the GSH/Cys
mixture divided by that of probe exposed to GSH only
(I(Cys+GSH)/I(GSH)), it was found that short incubation times
offered the highest apparent selectivity value
(I(Cys+GSH)/I(GSH)),35 Figure S3. In particular, probe incuba-
tion times in the 1–10 min range provided responses for
the combined 1 mM GSH and 100 ꢁM Cys solution that
are 1.5–2 times that of the 1 mM GSH solution alone,
pointing to a relatively good selectivity of DCM–Cys re-
sponse to Cys in the presence of cellularly relevant GSH
concentrations. To the best of our knowledge, these are the
first reported values that provide a direct and quantitative
assessment of probe selectivity for any cysteine-responsive
probe under conditions more reflective of those in respiring
mammalian cells.36
Figure 6. Fluorescence response (λex = 520 nm, λem = 640 nm)
of 30 ꢁM DCM-Cys probe incubated at 37 °C for 20 min with
150 ꢁM amino acids and potential interferents in 40%
DMSO:60% 0.1 M, pH 7.4 PBS. Bars represent % release of
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free DCM reporter with respect to that achieved for 20ꢀmin
incubation of DCM-Cys probe with 60 ꢁM Cys. N.B. interrupꢀ
tion in % DCM production axis.

Analytical Chemistry
Page 6 of 8
To further evaluate selectivity, the outcomes from ex-
As demonstrated by two-photon microscopy37 (Figure
7E), unadulterated H596 cells that were exposed to the
DCM–Cys probe for 10 min (N.B. 20 M probe is present
1
2
3
4
5
6
7
8
posure of DCM–Cys probe to other naturally-occurring
amino acids, such as glycine (Gly), methionine (Met),
serine (Ser), and threonine (Thr), were investigated. In
addition, species frequently encountered in biological
milieu, such as the ubiquitous ascorbic acid and NADH,
ꢁ
in solution in Figure 7) exhibited strong red fluorescence
in the 600–700 nm range, when compared to the cells
that were first incubated with the thiol scavenger NEM
(Figure 7F). In Figure 7D is displayed a representative
image of untreated H596 cells that was used as a control;
the lack of significant signal in the 600–700 nm range
indicates there were no other environmental interfer-
ences contributing to the observed fluorescence in the
DCM–Cys-treated cells, Figure 7E. The clear contrast in
fluorescence observed between images 7E and 7F indi-
cates the presence of cysteine in cells. Similar results
were observed upon inspection of single-photon confocal
microscopy images (Figure S5) of cells fixed after their
exposure to the probe or probe/thiol scavenger. We at-
tribute the quickly formed red fluorescence signal in
DCM–Cys-treated cells (10 min, Figure 7E) to the pres-
ence of DCM reporter that is produced upon entry of
electrically neutral DCM–Cys into cells (logarithm of the
octanol–water partition coefficient at pH 7.4 = 6.1; polar
surface area = 121 Å2)38,39 and its rapid reaction with intra-
cellular cysteine. These images further demonstrate the
multifunctional ability of the DCM–Cys probe as a single-
photon probe for fixed cell analysis, and importantly, as a
two-photon probe of cysteine presence in live cells. The
latter bodes well for imaging in tissues, as the penetration
depth will be greater both for light entering (900-nm ex-
citation) and exiting (640-nm emission) the field of exam-
ination. Further to such applications, the visible signal
from cells in Figure 7E, which was obtained in the presence
were also examined. A 30
Cys was incubated with 150
ferent at 37 °C for 20 min, and the fluorescence intensity
was measured ( ex = 520 nm, em = 640 nm). As shown in
ꢁ
M aqueous solution of DCM–
ꢁM of each potential inter-
9
λ
λ
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Figure 6, none of the non-thiol amino acids (Gly, Ser, and
Thr) nor the thiol-containing amino acid methionine
caused any significant increase in fluorescence. The bio-
logical reductant NADH, which is present at mM levels
within cells, and the common antioxidant ascorbic acid,
also did not cause release of the free reporter from the
DCM–Cys probe. A significant increase in fluorescence
was observed only upon addition of 60
ꢁM cysteine,
providing further evidence for the selective nature of the
reaction between the probe and cysteine. The results
from Figures 5 and 6 are readily verified by eye, as noted
by the visible color change under white light illumination
(Figure S4A) and the fluorescence color change under UV
lamp exposure (Figure S4B).
With the knowledge in hand that DCM–Cys allows for
selective in vitro detection of cysteine with low
probe–cysteine ratios and at relatively short incubation
time (1–10 min), the ability of DCM–Cys to detect cysteine
in live mammalian cells via two-photon microscopy and
traditional laser-scanning confocal microscopy was evalu-
ated using the human lung cancer cell line H596, Figures
7 and S5. The H596 cell line was chosen due to its known
non-protein thiol content,32 and its previous examination
with a class-selective thiol probe that responds to the
presence of Cys, GSH, and HCy.11
of 20 ꢁM DCM–Cys probe, is readily discernable from any
possible probe background fluorescence; this is the result
of the low fluorescence response from unreacted probe,
whose carefully designed structure leads to such behavior
(low absorptivity at the energy of excitation for the DCM
reporter and also low emission at the emission energy of
the reporter).
In order to provide initial work toward developing a
knowledge base concerning abilities of various probes to
sense cysteine presence in identical cell lines, we meas-
ured the signal-to-background ratio (SBR) achievable
with the DCM–Cys probe in living H596 cells. The SBR is
defined as the ratio of the integrated fluorescence intensi-
ties for living cells that are unadulterated/untreated (pos-
itive signal, 41 replicates) to that of NEM-treated cells
(background, 23 replicates). We determined the SBR to
be 4.4 for the DCM–Cys probe using the H596 cell line,
which, to our knowledge, is the first quantitative measure
of any cysteine-selective probe’s ability to sense cysteine
within mammalian cells. Typically, an SBR value above 1.5
is deemed to be significant with regard to application of
that probe in imaging studies.40
Figure 7. Microscopy images of living human H596 lung cells.
A–C,
differential
interference
contrast
images;
D–F, twoꢀphoton confocal microscope images (900ꢀnm excitaꢀ
tion, 600–700 nm emission). Images A and D, control experiꢀ
ment without probe; images B and E, incubation with
20 ꢁM DCM–Cys for 10 min at 37 °C; images C and F, expoꢀ
sure to 10 mM NEM thiol scavenger for 30 min at 37 °C, folꢀ
lowed by incubation with 20 ꢁM DCM-Cys and 10 mM NEM
for 10 min at 37 °C. Scale bar = 25 ꢁm.
ꢀ
CONCLUSIONS
We have successfully designed and synthesized a fluo-
rescent probe possessing a reactive acrylate moiety,
DCM-Cys, with the intention of selectively detecting cys-
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Page 7 of 8
Analytical Chemistry
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probe's response to Cys in simultaneous mixtures of Cys and GSH
with their concentrations reflective of those in mammalian cells, the
response of GSH alone was not reported under conditions similar to
that in the mixture of Cys and GSH, making it impossible to achieve
a direct comparison to the work with DCM-Cys. See: Liu, Y., Lv, X.,
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Supporting Information
Additional information as noted in text. Synthetic schemes,
characterization data for DCM–Cys and DCM (NMR and
Mass spectrometry), ESI/MS of cysteine-treated DCM–Cys
probe, visualization photos of probe interaction with cysteine
and potential cross-reactive species, fluorescence micro-
graphs of thiol selectivity within H596 cells. Supporting in-
formation is available free of charge via the Internet at
http://pubs.acs.org.
ꢀ AUTHOR INFORMATION
Corresponding Author
*Email: tunnel@LSU.edu. Fax: 225-578-3458; Tel: 225-578-
3239.
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ꢀ
ACKNOWLEDGMENTS
This material is based upon work supported by the US Na-
tional Science Foundation under grant CHE-1507975. We
extend our appreciation to the LSU AgCenter Biotechnology
Laboratory Animal Cell Culture Facility for use of infrastruc-
ture and instrumentation. This work utilized the facilities of
the Cell Biology and Bioimaging Core that are supported in
part by COBRE (NIH 8 P20-GM103528) and NORC (NIH
2P30-DK072476) center grants from the National Institutes
of Health. B.P. thanks the Louisiana Economic Development
Assistantship Program at LSU.
ꢀ
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For ToC use only.
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