A FRET-based two-photon probe for: In vivo tracking of pH during a traumatic brain injury process

Article abstract of DOI:10.1039/c9nj04049h

The traumatic brain injury (TBI) process, a significant cause of neurodegenerative diseases, is generally thought to be accompanied by a decrease in pH of the intracellular environment. In this work, a fluorescence resonance energy transfer (FRET)-based ratiometric two-photon fluorescent pH probe, FRET-pH, has been designed and fully characterized to monitor the pH change so as to understand the extent of the TBI process. FRET-pH, which has nearly 100% energy transfer efficiency and ～100-fold ratio signal changes, is constructed from a naphthalene derivative ADN as a two-photon fluorophore and SRhB as a recognition domain for pH. FRET-pH displays both one- A nd two-photon ratiometric responses towards pH in aqueous solution. The appropriate pK_a value (5.89) supports its use in subsequent biological applications. Finally, two-photon microscopic imaging of pH in living HeLa cells, BV-2 cells and rat brain tissue has been achieved.

Full text of DOI:10.1039/c9nj04049h

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A FRET-based two-photon probe for in vivo
tracking of pH during a traumatic brain injury
Cite this: New J. Chem., 2019,
process†
43, 17018
a
b
a
a
b
Baoping Zhai,* Shuyang Zhai, Ruilin Hao, Jianjun Xu and Zhihong Liu
*
The traumatic brain injury (TBI) process, a significant cause of neurodegenerative diseases, is generally
thought to be accompanied by a decrease in pH of the intracellular environment. In this work, a
fluorescence resonance energy transfer (FRET)-based ratiometric two-photon fluorescent pH probe,
FRET-pH, has been designed and fully characterized to monitor the pH change so as to understand the
extent of the TBI process. FRET-pH, which has nearly 100% energy transfer efficiency and B100-fold
ratio signal changes, is constructed from a naphthalene derivative ADN as a two-photon fluorophore
and SRhB as a recognition domain for pH. FRET-pH displays both one- and two-photon ratiometric
Received 4th August 2019,
Accepted 8th October 2019
a
responses towards pH in aqueous solution. The appropriate pK value (5.89) supports its use in
DOI: 10.1039/c9nj04049h
subsequent biological applications. Finally, two-photon microscopic imaging of pH in living HeLa cells,
BV-2 cells and rat brain tissue has been achieved.
rsc.li/njc
well known that single fluorescence emission intensity may be
affected by many factors, such as the intensity of the light source,
Introduction
Severe traumatic brain injury (TBI) caused by external mechanical the sensitivity of the sensor, the scattering and viscosity of the
concussion, falls, traffic accidents, etc., will become the leading solvent, and more importantly, the distribution and concentration
1–4
16
risk factor of mortality and disability by the year 2020. Research of the probe molecules. In contrast, a ratio-based fluorescent
has shown that TBI is a significant cause of neurodegenerative probe could simultaneously present two different emissions.
diseases, and it is usually associated with prolonged and progres- Recording the ratio signals at different wavelengths as detection
5
–9
sive neuroinflammation.
Literature reports have shown that signals allows for a built-in correction of the above factors, so as to
17
the TBI process is usually accompanied by inflammatory reac- solve these problems and achieve more accurate analysis. So far,
tions that increase oxygen consumption and cause BV-2 cells to be a large number of studies based on ratio fluorescent probes have
in a hypoxic state. Cellular anaerobic respiration leads to a sharp been reported. Intramolecular charge transfer (ICT) and fluores-
10,11
decrease in mitochondrial pH.
Thus, it is of urgent impor- cence resonance energy transfer (FRET) are the two most com-
tance to develop a reliable tool to achieve real-time monitoring of monly used sensing mechanisms for designing ratio fluorescence
18
pH change, to understand the extent of neurological damage and probes. However, some ICT-based ratio fluorescent probes have
12,13
to seek opportunities for novel strategies of TBI treatment.
very wide emission spectra, which often result in serious overlap
With the development of imaging techniques, small mole- of the probe signals after target analyte recognition, leading to
19
cule fluorescent probes provide powerful tools for the real-time unreliability of the detection results. On the other hand, reactive
detection and visualization of certain targets both in in vitro probes usually have a strong ICT effect and can achieve larger
and in vivo biological applications owing to their non-invasive pseudo-Stocks shifts. Nevertheless, the change of molecular con-
treatment, structural and optical modularity, high sensitivity, jugate structure during the reaction makes it difficult to realize
1
4,15
and high temporal and spatial resolution.
However, it is single-wavelength excitation (including one- and two-photon
excitation), which causes great complexity and unreliability in the
20–29
a
detection.
Department of Chemistry, Xinzhou Teachers University, Xinzhou, Shanxi, 034000,
China. E-mail: bpzhai@aliyun.com
FRET is a non-radiative process in which a dye donor in the
b
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of
Education), College of Chemistry and Molecular Sciences, Wuhan University,
Wuhan 430072, China. E-mail: zhhliu@whu.edu.cn
excited state transfers energy to a dye acceptor in the ground
30–32
state through long-range dipole–dipole interactions.
In
addition, probes based on the FRET mechanism can produce
†
Electronic supplementary information (ESI) available: Synthesis and character-
larger pseudo-Stokes shifts, thus avoiding the scattering effect
caused by molecule excitation. In order to obtain a strong
ization of SRhB, 7, and FRET-pH and supplementary spectral results. See DOI:
3
3
1
0.1039/c9nj04049h
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ratio signal and increase the sensitivity and reliability of probe
detection, ratiometric probes based on FRET always possess
properties including: (1) adequate spectral overlap between the
emission peak of the donor and the excitation peak of the
acceptor to achieve greater energy transfer efficiency; (2) larger
spectral separation between two emissions of the donor and the
acceptor to guarantee high accuracy in the measurement of
fluorescence intensity ratios; (3) comparable brightness (e  F)
of the donor fluorophore and the acceptor dye (which may
increase the sensitivity of the probe to target response through
3
3
the larger ratio signal changes).
Based on the above considerations, we designed and synthe-
sized a two-photon excited ratiometric pH probe, FRET-pH,
based on the FRET mechanism. Two-photon microscopy
(
(
TPM), excited by two photons in the near-infrared window
700–1100 nm), provides solutions to several problems arising
Fig. 1 (A) Two-photon active cross section (Fd) of ADN under 780–850 nm
excitation light; (B) absorption of SRhB (red line) and the emission of ADN (black
from one-photon microscopy (OPM), such as short excitation
wavelength (350–550 nm), photo-bleaching, light damage and line). (C) Fluorescence and (D) two-photon fluorescence spectra of FRET-pH
3
4–46
(10 mM) in different pH buffers: pH = 7.4 (black line), pH = 4.6 (red line).
auto-fluorescence from biomolecules.
To this end, we
synthesized a molecule using the naphthalene derivative
ADN with good two-photon properties as an energy donor
3 3
and SRhB as an energy acceptor to identify the pH through an (ESI†), we used DMF, DMSO, CH CN, CH OH and EtOH and found
fl
open–closed mechanism. The probe FRET-pH showed a high that the quantum yield in ethanol is up to 0.76 (lmax = 454 nm). At
abs
energy-transfer efficiency of up to nearly 100%. As the pH the same time, the absorption peak at a wavelength (lmax) of
5
À1
À1
decreases, the ratio signal increases by B100-fold. Finally, we 350 nm (e = 1.17 Â 10 M cm ) was also observed. To simulate
applied it to the model of traumatic brain injured mice and the biological environment, we used 10% EtOH in PBS buffer
explored the relationship between traumatic brain injury and solution and excellent photophysical properties were still being
abs
neuroinflammation.
demonstrated with an absorption peak at a wavelength (l ) of
max
5
À1
À1
3
(l
65 nm (e = 1.15 Â 10 M cm ) and the fluorescence maximum
fl
max) at 400 nm (F = 0.45). What’s more, the two-photon active
À50
3
Results and discussion
Preparation and photophysical properties of the probe
cross section reached 60 GM (1 GM = 1 Â 10
cm s per photon)
at 810 nm, as shown in Fig. 1A. On the other hand, SRhB has a
very good response to pH (Fig. S9, ESI†), and the absorption
spectrum of SRhB is perfectly matched to the fluorescence
emission spectrum of the energy donor ADN, as shown in
Fig. 1B. Therefore, we chose SRhB as both the energy acceptor
and the pH response group. The synthetic methodology for ADN,
SRhB and the probe FRET-pH are outlined in Scheme S1 (ESI†),
To achieve a reasonable probe with better detection sensitivity
and increase the signal-to-background ratio, we first constructed
a high energy transfer probe via two-photon energy resonance
transfer (TP-FRET). A large number of literature reports indicate
that the TP-FRET system requires an energy donor with good
two-photon activity, and an energy acceptor that has a strong
enough fluorescence emission intensity and sufficient spectral
match with the donor. For this purpose, a two-photon pH probe
using 6-hydroxyl-quinonline-2-benzothiazole (ADN) as the fluoro-
phore and a derivative of Rh6G (SRhB) as a recognition domain
for the detection of pH (FRET-pH) was successfully synthesized
1
13
and their structures were confirmed by H NMR, C NMR and
HRMS (see Fig. S1–S7, ESI†).
Before the spectral detection, we first explored the solubility
of the probe. The water solubility of the probe FRET-pH was
measured in PBS buffer at pH 7.4. As shown in Fig. S10 (ESI†),
the UV absorption was still in a linear range at a probe
concentration of 10 mM, indicating that the condition can be
used for solution and cell detection. It has been shown in the
literature that the pH environment of a lipid-soluble probe is
(Scheme 1). The energy donor ADN has excellent photophysical
properties in different solvents. As shown in Fig. S8 and Table S1
4
7
greatly affected by the surfactants in the solution, so it is
necessary to evaluate the effects of different surfactants (Triton,
CTAB and SDS) on the probe response. The results show that
the probe FRET-pH was unable to respond to pH under the
conditions of Triton and CTAB, and had a good pH response
only in the presence of SDS, as shown in Fig. S11 (ESI†). To this
end, in the presence of 25 mM SDS in PBS buffer, we used the
one-photon and two-photon modes respectively to probe the
fluorescence response (as shown in Fig. 1C and D). Under
Scheme 1 Structure and response mechanism of FRET-pH to pH.
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neutral conditions (pH = 7.4), FRET-pH shows the emission concentration of 0.1 mM. In addition, in an acidic environment
peak at 501 nm. When the environmental acidity changes from (pH = 4.6), the fluorescence signal of the reaction system could
neutral to acidic, a new fluorescence peak emerges with gradu- reach a platform in about 2 min. Meanwhile, there was no
ally increasing intensity at 562 nm, which presents a 61 nm fluorescence intensity ratiometric change that could be observed
red-shift as compared to that at pH 7.4. In addition, the probe under the neutral condition (pH = 7.4) (Fig. S13, ESI†), demon-
has nearly 100% energy transfer efficiency, and a B100-fold strating that FRET-pH is stable in the reaction system.
fluorescence intensity ratio enhancement at 562/501 nm when
present in different pH environments. Compared with the Two-photon microscopic imaging of pH in BV-2 cells
reported pH probes, FRET-pH has better sensitivity, greatly
Before utilizing FRET-pH in bioimaging, the cytotoxicity was
increases the signal-to-background ratio and can be used in
examined by methyl thiazolyl tetrazolium (MTT) assay. As shown
the bioimaging process. Therefore, we use this system for
in Fig. S14 (ESI†), more than 90% cells could survive after being
incubated with FRET-pH (10 mM) for 24 h. Subsequently, the
subsequent research studies.
photostability of FRET-pH was tested by illuminating the probe-
loaded cells for 30 min. We traced the fluorescence intensity of
Sensing performance of pH
The sensing performance of FRET-pH towards pH in phosphate the red channel (540–650 nm). As shown in Fig. S15 (ESI†), a
buffer (10 mM, 0.9% NaCl in the presence of 25 mM SDS) was negligible decrease can be observed under two-photon excitation,
investigated. The fluorescence titration of FRET-pH (Fig. 2A) which makes observation in living organisms possible for a long
shows that the probe is highly sensitive in the pH range of time period. Therefore, FRET-pH was utilized to image pH in
4
.6–7.4. The titration curve (Fig. 2B, red line) exhibits distinct BV-2 cells that were incubated with 10 mM FRET-pH for 30 min for
pH-dependent fluorescence changes in this range. In contrast, the probe loading, and then treated with pH 4.6, 5.0, 5.4, 5.8, 6.6 and
+
+
titration curve of ADN exhibits no significant variation, which 7.0 universal buffers containing K /H ionophores (nigericin and
34
indicates that the energy donor has distinct pH-independent monensin) for 20 min. Fluorescence emission was collected at
fluorescence changes (Fig. 2B and Fig. S12, ESI†). The pK
of 450–530 nm (green emission channel) and 540–650 nm (red
a
the probe is calculated to be 5.89 Æ 0.03 according to the titration emission channel), respectively. The cells loaded with FRET-pH
curve (Fig. 2C), which is ideal for the detection of traumatic brain in pH 7.0 universal buffer present rather weak ratio fluorescence
injury (acidic environment). The selectivity of FRET-pH probe and an obvious green fluorescence (Fig. 3f and Fig. S16, ESI†).
+
towards pH was evaluated by using some biological interference With increasing H concentration, the intensity of red fluores-
ions as potential interferents. As shown in Fig. 2D, for detection at cence and ratio fluorescence are gradually enhanced (Fig. 3a–d
both pH 4.6 and 7.4, no significant variation can be observed in and Fig. S16, ESI†), which is consistent with the above solution-
the presence of a variety of ions and small molecules at the phase results. The distinct discrimination of signals at pH 4.6–7.0
confirms the ability of FRET-pH to sensitively detect the extent of
traumatic brain injury. Moreover, the relative fluorescence inten-
sity ratio of the two channels (Ired/Igreen) can provide quantitative
information for estimating the intracellular pH value (Fig. 3g).
To further understand the pH changing in the traumatic
brain injury (TBI) process, we first established a microglial injury
model using lipopolysaccharide (LPS)-mediated chemical brain
injury. It has been shown that microglia and astrocytes play
critical roles in initiating neuroinflammation when injury
occurs, and that the inflammatory process is accompanied by
a decrease in pH of the intracellular environment. Therefore, we
try to use the probe FRET-pH to monitor the change of pH in the
Fig. 2 (A) Fluorescence spectra of FRET-pH (10 mM) at acidic to neutral pH
values (4.0–7.4) and (B) the titration curve of FRET-pH and ADN. (C) Plot of
pH vs. log[(Imax À I)/(I À Imin)] using the Henderson–Hasselbach type mass
action equation; log[(Imax À I)/(I À Imin)] = pK À pH, where I is the observed
a
fluorescence intensity ratio of FRET-pH. (D) Responses of 10 mM FRET-pH to
+
+
2+
2+
2+
0
.1 mM various species: (a) control, (b) Na , (c) K , (d) Fe , (e) Cu , (f) Ca
,
Fig. 3 (a–f) Ratiometric TPM images (Ired/Igreen) of ionophore-treated
BV-2 cells incubated with 10 mM FRET-pH at pH 4.6, 5.0, 5.4, 5.8, 6.6
and 7.0. (b) Two-photon Ired/Igreen titration with pH in BV-2 cells. Excitation
wavelength is 810 nm. Scale bar: 20 mm. Cells shown are representative
images from replicate experiments (n = 10).
2+
3+
3+
+
À
À
(g) Zn , (h) Al , (i) Fe , (j) Cu , (k) H
2
O
2
, (l) ONOO , (m) ClO , (n) KO
À
2
, (r) NO , (s) NaHS, (t) Na , (u) NaHSO (v) Cys, (w)
2
,
ꢀ
1
(o) OH, (p) NO, (q) O
2
2
S
n
3
GSH and (x) HCy. The above experiments were done in PBS buffer (10 mM,
pH 7.4, containing 0.9% NaCl and 25 mM SDS).
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À1
Fig. 4 (a–f) TPM of BV-2 cells pre-treated with 0, 10 and 20 mg mL LPS
for 14 hours before incubation with 10 mM FRET-pH for 30 min. Images
were obtained by being excited with 810 nm laser light, and the emissions
were collected individually in the red channel (540–650 nm, a–c) and
the green channel (450–530 nm, d–f). (g–i) Ratiometric TPM images
Fig. 5 Rat brain tissue TPM images pretreated with 200 mL of probe
FRET-pH (1.0 mM) (a and b) without and (c and d) with brain injury in 4
day. Images were obtained by being excited with 810 nm laser light, and
the emissions were collected individually in the red channel (540–650 nm,
a–c) and the green channel (450–530 nm, d–f). (e and f) Ratiometric TPM
images (Ired/Igreen) of (a–d). (g) Mean intensities of (e and f). Scale bar:
200 mm. The data were shown as mean (Æs.d.) (n = 3). Difference was
analyzed by one-way ANOVA and Bonferroni post-test. *P o 0.05,
(Ired/Igreen) of (a–f). (j) Mean intensities of (a–i). The data were shown as
mean (Æs.d.) (n = 7). Difference was analyzed by one-way ANOVA and
Bonferroni post-test. *P o 0.05, **P o 0.01 or ***P o 0.001 vs. blank
group. Scale bar: 20 mm.
**P o 0.01 or ***P o 0.001 vs. blank group.
TBI process using the two-photon microscope. We therefore
selected this cell model for pH observations. As shown in
Fig. 4g, BV-2 cells treated with only FRET-pH (10 mM) showed
moderate ratio fluorescence, which could have originated from
the probe itself and the intrinsic basal pH. When the cells were
Conclusions
In conclusion, by aiming at a high sensitivity with enhanced
ratiometric two-photon fluorescence pH detection, we have
designed and synthesized a novel TP fluorescence probe,
FRET-pH, for ratiometric sensing. This probe can rapidly
respond and offer high selectivity and sensitivity in monitoring
À1
pre-treated with 10 and 20 mg mL LPS before the loading of
probe, a remarkable enhancement of fluorescence ratio (Ired/Igreen
)
was observed (Fig. 4h and i). These results unambiguously con-
firmed that the FRET-pH probe is capable of monitoring the pH
changing during the LPS-mediated TBI process in BV-2 cells.
+
H in BV-2 cells. The probe exhibits both one-photon and two-
photon excited remarkable fluorescence enhancement for pH.
It also shows a high selectivity to pH among many interfering
species. Owing to the excellent sensing performance and two-
Two-photon microscopic imaging of pH in rat brain tissue
In order for a molecular probe to possibly cross the blood–brain
barrier (BBB), its lipophilicity index log P normally needs to be
within the range of 1.0–3.0. The log P value of FRET-pH was
calculated as 1.63, fulfilling the criterion of lipophilicity. Dur-
ing the TBI process, the injured brain tissues can recruit BV-2
cells to remove apoptotic cells and toxic substance and produce
+
photon excitation nature, the probe successfully monitors H in
the traumatic brain injury (TBI) process. The results of this
work suggest that the developed probe may find applications in
in vivo pH detection, which will help us to understand more
deeply the TBI process.
+
10,11
a high local concentration of H .
By using the z-scan mode
of the TP microscope, we first investigated the fluorescence
intensities at different depths of rat brain tissue (Fig. S17, ESI†).
The accumulated images obtained with z-scan reveal that FRET-
Conflicts of interest
pH can homogeneously stain the tissues, and that it is able to The authors declare no competing financial interest.
visualize at depths of up to 140 mm in rat brain tissues under TP
excitation. Motivated by the deep imaging depth in tissues, we
attempted to probe pH in rat brain tissue by two-photon
microscopy during the TBI process. As shown in Fig. 5, the
Acknowledgements
fluorescent images in the brain were collected from two This work has been financially supported by the Key Disciplines
fluorescence channels: red channel (Fred, emission wavelength: Construction Foundation of Xinzhou Teachers University
540–650 nm) and blue channel (Fgreen, emission wavelength: (XK201305) and the National Natural Science Foundation of
450–530 nm). Compared to the treated group, the fluorescence China (21575109). All animal procedures were performed in
ratio (red/green) of the injured mice displayed a significant accordance with the Guidelines for Care and Use of Laboratory
+
increase, indicating that H was overproduced in the brain Animals of Hunan University, and experiments were approved by
injury process (Fig. 5e and f). The ratio of the average fluores- the Animal Ethics Committee of the College of Biology (Hunan
cence intensity (Fred/Fgreen) in Fig. 5(a–f) is displayed in Fig. 5g. University).
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