A two-photon turn-on probe for glucose uptake

Article abstract of DOI:10.1039/c2cc16792a

We report a two-photon turn-on probe (AS1) that can be excited by 780 nm femto-second pulses and visualize glucose uptake and the changes in the intracellular glucose concentration in live cells and tissue by two-photon microscopy.

Full text of DOI:10.1039/c2cc16792a

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A two-photon turn-on probe for glucose uptakew
a
b
Chang Su Lim,z Chul Chung,z Hwan Myung Kim,b Myung Jin An,a Yu Shun Tian,c
Hoon Jai Chund and Bong Rae Cho*a
Received 2nd November 2011, Accepted 22nd December 2011
DOI: 10.1039/c2cc16792a
We report a two-photon turn-on probe (AS1) that can be excited
by 780 nm femto-second pulses and visualize glucose uptake and
the changes in the intracellular glucose concentration in live cells
and tissue by two-photon microscopy.
Glucose plays vital roles in living systems. Glucose homeostasis
is closely associated with energy metabolism and modulated
primarily by the insulin-mediated signaling pathway.1 Defects
in insulin secretion or insulin action may result in abnormally
Scheme 1 Synthesis of AS1.
high levels of blood glucose,2 which can lead to type-2 diabetes,
metabolic syndrome, hypertension, and increased cardio-
vascular risk.3 In this context, a key challenge for elucidating
the biological roles of glucose in living systems is the ability to
monitor glucose at the cell, tissue, and organism level. For this
purpose, a variety of fluorescent probes derived from anthra-
cence, pyrene, and stilbene as the fluorophore and boronic
acid as the binding site for the monosaccharides have been
developed.4,5 However, use of these probes with one-photon
(OP) microscopy requires relatively short excitation wave-
lengths (B350–500 nm), which limits their use in deep tissue
imaging due to the shallow depth of penetration (o80 mm).
Except for in vivo cancer diagnosis by positron emission tomo-
graphy (PET) using [18F]-2-fluoro-2-deoxyglucose (18FDG),6
methods for imaging glucose in thicker samples, such as tissues
and whole organisms, are relatively rare.
for more than 3000 s with successful application in the
therapeutic efficacy of anticancer agents.9 However, AG2 is
not suitable for the real-time monitoring of glucose because it
can detect the rate of uptake only, but not that of decrease. To
monitor both processes by TPM, it is crucial to develop a TP
turn-on probe for glucose. However, there has been no report
on such probes.
The requirements for a TP turn-on probe to visualize
glucose uptake include a large enhancement in the TP excited
fluorescence (TPEF) intensity upon binding with glucose,
sufficient water solubility for staining cells and tissues, pH
resistance, and high photostability.10,11 Our strategy was to
link the boronic acid moeity with the 2-acetyl-6-dimethyl-
aminonaphthalene (acedan) through a glycinamide linkage
(AS1, Scheme 1). Acedan is a polarity-sensitive fluorophore
that has been successfully employed in a number of TP
probes,10,11 and phenylboronic acid is a well-known receptor
for monosaccharaides.4,5 We now report that AS1 can monitor
glucose uptake in live cells and tissues.
An attractive approach for the detection of glucose deep
inside living tissues is the use of two-photon microscopy
(TPM).7,8 TPM, which utilizes two photons of lower energy
for the excitation, is becoming increasingly more popular in
biomedical research due to the advantages it offers, which
include increased penetration depth (>500 mm), localized
excitation, and prolonged observation time.7,8 Recently, we
developed a TP glucose tracer, AG2, which can monitor glucose
uptake in normal and colon-cancer tissues from human patients
AS1 was prepared in 31% yield by the reaction of A with
AH1 as shown in Scheme 1 (See ESIw for details). The
solubility of AS1 in PBS buffer solution (30 mM, pH 7.2,
I = 0.10) is B3.0 mM (Fig. S1, ESIw), which is sufficient for
staining the cells.
The absorption and emission spectra of AS1 showed gradual
bathochromic shifts with the solvent polarity (ENT )12 in the order
1,4-dioxane o DMF o EtOH o H2O (Fig. S2 and Table S1,
ESIw). The large bathochromic shift for the emission (B80 nm)
spectra indicates the utility of AS1 as a polarity probe.
When D-glucose was added to AS1 in phosphate-buffered
saline (PBS, pH 7.4), the fluorescence intensity increased
gradually without affecting the absorption spectra presumably
because of the blocking of the photo induced electron transfer
(PeT) by complexation with D-glucose (Fig. 1a and 4S).13
a Department of Chemistry, Korea University, 145-Anam-ro,
Sungbuk-ku, Seoul, 136-713, Korea. E-mail: chobr@korea.ac.kr;
Fax: +82-2-3290-3544
b Division of Energy Systems Research, Ajou University, Suwon,
443-749, Korea
c College of Pharmacy, Yanbian University, Yanji, Jilin, P. R. China
d Department of Internal Medicine, College of Medicine,
Korea University, 1-Anamdong, Seoul, 136-705, Korea
w Electronic supplementary information (ESI) available: Experimental
details for the synthesis, photophysical studies, and cell and tissue
imaging. See DOI: 10.1039/c2cc16792a
z These two authors contributed equally to this work.
c
2122 Chem. Commun., 2012, 48, 2122–2124
This journal is The Royal Society of Chemistry 2012

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other acedan-derived TP probes.10,11 This predicts a bright
TPM image of the samples stained with AS1.
We then evaluated the ability of AS1 to detect mono-
saccharides in live cells (Fig. 2). Upon TP excitation at 780 nm,
the TPM image of the HeLa cells labeled with 2 mM of AS1 for
20 min at 37 1C shows appreciable background emission
(Fig. 2a). Because the fluorescence intensity of AS1 increases
at pH o 5 (Fig. S3b, ESIw), there is a possibility that the
probes in the lysosomes might partially contribute to the TPM
images. To assess such a possibility, HeLa and primary cortical
neuronal cells were co-stained with AS1 and LysoTracker Red
(LTR), a well-known OP fluorescent probe for acidic vesicles,17
and the images were co-localized. The TPM image of AS1
showed partial overlap with the OP microscopy image of LTR
(Fig. S7c and S7f, ESIw). Pearson’s colocalization coefficient,
A, which describes the correlation of the intensity distribution
between the channels,18 was calculated by using Autoquant X2
software. The A values of AS1 with LTR for HeLa and
primary cortical neuronal cells were 0.21 and 0.16, respec-
tively, indicating that AS1 exists primarily in the cytoplasm
with a small fraction in the lysosomes. Moreover, this effect
would not influence the TPEF intensity changes shown in
Fig. 2g and h because addition of D-glucose is not expected to
change the AS1 concentration in the lysosome (vide infra).
When 20 mM of D-glucose was added to the cells, the TPEF
increased abruptly and then decreased to the base level (Fig. 2c).
Fig. 1 (a) One-photon emission spectra of AS1 in PBS (pH 7.4) in the
presence of D-glucose (0–1.0 M). (b) One- (black) and two-photon
(white) fluorescence titration curves for the complexation of AS1 with
D-fructose (’,&), D-galactose (m,n), and D-glucose (K,J) (0–1.0 M).
The excitation wavelengths for one- and two-photon processes were
365 and 780 nm, respectively.
The fluorescence enhancement factors [FEF = (F ꢀ Fmin)/Fmin
]
calculated for the one- and two-photon (TP) processes were
4.0 in the presence of 1.0 M D-glucose (Table 1).
The dissociation constants (KOd P) for the binding of AS1
with sugars calculated from the fluorescence titration curves
measured in PBS (pH 7.4) are KOd P = 0.60 ꢁ 0.03, 0.35 ꢁ 0.01,
and 0.048 ꢁ 0.002 M for D-glucose, D-galactose, and
D-fructose, respectively.14,15 The corresponding values measured
by TP processes are 0.62 ꢁ 0.04, 0.38 ꢁ 0.02, and 0.050 ꢁ 0.004 M,
respectively (Table 1, Fig. 1b, S4, and S5, ESIw), indicating
that AS1 can detect these sugars in the sub-molar range by
TPM. Moreover, the titration curve fitted well with the 1 : 1
binding model and the Hill plots were linear with a slope of
1.0, indicating 1 : 1 complexation between the probe and the
sugars (Fig. S4c, S5c, and S5f, ESIw).14,15 The pKa value of
AS1 was 5.3, which is larger than 4.5 reported for the parent
compound without the boronic acid moiety (AH1) (Fig. S3b,
ESIw), presumably because of the less electronegative nature of
boron than carbon. Moreover, the fluorescence of AS1 was
pH-insensitive at pH > 6 (Fig. S3b, ESIw). Hence, this probe
is useful to detect monosaccharides in the biologically relevant
pH range.
The decrease could be fitted to a first order decay with t1/2
=
170 s, indicating that the intracellular D-glucose concentration is
decreased at this rate (Fig. S8, ESIw). A similar result was
observed with primary cortical neuronal cells (Fig. 2d–f, h and S8,
ESIw). In contrast, no change was observed with D-galactose
and D-fructose, presumably because D-glucose is the primary
energy source for the cells (Fig. 2 and S9, ESIw). Moreover, the
TP excited fluorescence (TPEF) intensity at a given spot on the
AS1-labeled HeLa cells remained nearly the same after continu-
ous irradiation of the fs-pulses for 60 min, indicating high
photostability (Fig. S10, ESIw). Further, AS1 shows negligible
toxicity over the course of imaging experiments as measured
by using a CCK-8 kit (Fig. S11, ESIw). These data establish
that AS1 can monitor D-glucose uptake and the changes in
the intracellular glucose concentration in the live cells with
minimum intereference from pH, other monosaccharides,
photostability, and cytotoxicity.
The two-photon cross section (d) of AS1 in PBS (pH 7.4)
was measured in the presence of excess D-glucose by the TPEF
measurement technique using the femto-second (fs) laser
pulses as described.16 In all cases, the output intensity of TPEF
showed quadratic dependency on the input laser intensity,
thereby confirming the occurrence of nonlinear absorption
(Fig. S6b, ESIw). The TP action spectra of AS1 in the presence
of excess D-glucose in the PBS buffer indicated a Fd value of
85 GM at 780 nm (Fig. S6a, ESIw), which is similar to those of
We next sought to apply AS1 in tissue imaging. The
hippocampus was isolated from a 2 day old rat, and a slice
was incubated with 20 mM AS1 for 1 h at 37 1C. Since it takes
Table 1 Photophysical data for AS1
b
Compounda
l(m1a) x/lflmax
Fc
KOd P d/M
FEFe
Fdf
AS1
366/500
365/498
365/498
365/498
0.040
0.51
0.24
0.16
ndg
AS1–D-fructose
AS1–D-galactose
AS1–D-glucose
0.048 ꢁ 0.002 (0.050 ꢁ 0.004)h
0.35 ꢁ 0.01 (0.38 ꢁ 0.02)h
0.60 ꢁ 0.03 (0.62 ꢁ 0.04)h
12 (13)h
5 (6)h
4 (4)h
85
a
b
All data were measured in PBS (pH 7.4) in the absence and presence (1.0 M) of D-fructose, D-galactose, and D-glucose.
d
lmax of the one-photon
c
absorption and emission spectra in nm. Fluorescence quantum yield, ꢁ15%. Dissociation constants for D-fructose, D-galactose, and D-glucose
e
f
measured by one-photon processes, ꢁ10%. Fluorescence enhancement factor, (F ꢀ Fmin)/Fmin, measured by one-photon processes. The peak
two-photon action cross section in 10ꢀ50 cm4 s/photon (GM), ꢁ15%. The two-photon excited fluorescence intensity was too weak to measure the
g
h
cross section accurately. The numbers in the parentheses are measured by the two-photon process.
c
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GLUT3 vesicles with the plasma membrane,20 the TPEF
intensity increased (Fig. 3e). This indicates D-glucose uptake
by the hippocampal slice. Here again, the uptake of D-fructose
and D-galactose was not detected in any of these regions
(Fig. S14, ESIw). Therefore, AS1 is clearly capable of detecting
the D-glucose uptake in live tissues by TPEF response.
To conclude, we have developed a TP probe (AS1), which
shows a significant TP cross section, of 4–12-fold TPEF
enhancements in response to sugars and dissociation constants
(KTdP) of (0.50 ꢁ 0.04)–(0.60 ꢁ 0.03) M. This probe can detect
glucose uptake and the changes in the intracellular glucose
concentration in live cells and living tissues. We expect that
this probe will find useful applications in biomedical research.
This work was supported by a grant of the Korea Health-
care technology R & D Project, Ministry of Health & Welfare,
Republic of Korea (A111182).
Notes and references
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Fig. 2 Pseudocolored TPM images of 2 mM AS1-labeled (a–c) HeLa
cells and (d–f) primary cortical neuronal cells at (a, d) 100, (b, e) 300,
and (c, f) 1000 s. (g, h) The changes of the TPEF intensity of
AS1-labeled (g) HeLa cells and (h) primary cortical neuronal cells as
a function of time. 20 mM of D-glucose, D-fructose, and D-galactose
were added after 200 s. The TPEF were collected at 500–620 nm upon
excitation at 780 nm with a fs pulse. Scale bar, 30 mm. Cells shown are
representative images from replicate experiments (n = 3).
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Fig. 3 (a–c) TPM images of a fresh rat hippocampal slice stained with
20 mM AS1 at a depth of ca. 100 mm with a magnification of 10ꢂ. TPM
images were obtained at (a) 100, (b) 1500, and (c) 3000 s. After 200 s, the
tissue was treated with 50 mM D-glucose, 50 nM insulin, and 50 mM
KCl. (d) Bright field image. (e) Time course of TPEF intensity at
designated positions 1–3 in (a). The TPEF were collected at 500–620 nm
upon excitation at 780 nm with a femtosecond pulse. Scale bar, 300 mm.
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longer time to stain the tissues during which they may be
deformed, excess amount (20 mM) of AS1 was used to facil-
itate staining. A bright field image of this slice reveals the CA1
and CA3 regions and the dentate gyrus (DG; Fig. 3d and
S12, ESIw). The TPM images acquired at 80–150 mm depth
(Fig. S13, ESIw) reveal the basal sugar levels in these regions.
When 50 mM D-glucose was added to the tissue pretreated
with 50 nM insulin, which transports a glucose transporter
GLUT3 to the plasma membrane,19 and 50 mM KCl
(Fig. 3a–c), a membrane depolarizer that induces fusion of
c
2124 Chem. Commun., 2012, 48, 2122–2124
This journal is The Royal Society of Chemistry 2012