Construction of a fluorescent probe for selectively detecting singlet oxygen with a high sensitivity and large concentration range based on a two-step cascade sensing reaction

Article abstract of DOI:10.1039/c9cc04300d

A novel fluorescent probe XQ-1 for selectively detecting ¹O₂ on the basis of a two-step cascade reaction has been rationally constructed. The probe responded to ¹O₂ not only showing a high sensitivity, but also displaying a large concentration range, which means that the probe can be used as a powerful tool to monitor the efficacy of PDT toward cancer and concurrently track the adverse effects on healthy cells.

Full text of DOI:10.1039/c9cc04300d

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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Construction of fluorescent probe for selectively detecting singlet
oxygen with high sensitivity and large concentration range based
on two-step cascade sensing reaction
Received 00th January 20xx,
Accepted 00th January 20xx
Lingliang Long,*a Xiangqi Yuan,a Siyu Cao,a Yuanyuan Han,a Weiguo Liu,a Qian Chen,a Aihua Gong,
*b and Kun Wang*a,c
DOI: 10.1039/x0xx00000x
A novel fluorescent probe XQ-1 for selectively detecting 1O2 on the
basis of two-step cascade reaction has been rationally constructed.
The probe responded to 1O2 not only showing high sensitivity, but
also displaying large concentration range, which renders the probe
can be used as a powerful tool to monitor the efficacy of PDT to
cancer and concurrently track the adverse effect to healthy cells.
fluorescent probes with fluorescein, rhodamine and europium
complex as fluorophore and 9,10-disubstituted anthracene as 1O2
trap have been developed.6 Very few fluorescent probes using furan7
and histidine8 as 1O2 trap were also constructed. Zhang et al designed
mitochondria targeted probe for detecting 1O2 during the PDT
process.9 Moreover, chemiluminescence probes with enol-ether or
1
anthracene as O2 trap were judiciously developed.10 Nonetheless,
1
these reported probes for detection of O2 during the PDT process
Singlet oxygen (1O2) is a selectively reactive oxygen species (ROS) and
plays significant roles in a variety of physical, chemical, biological,
and medical events.1 Particularly, the 1O2 is appealing in recent
medical studies due to it exerts main effect on photodynamic
therapy (PDT), a minimally invasive methodology for anticancer
treatment.2 In the process of PDT treatment, upon selective
photoirradiation of the cancer region, the non-toxic photosensitizers
(PS) and oxygen cause the production of 1O2 that can give a cytotoxic
activity toward cancer cells.3 Nevertheless, the PDT is essentially an
were still retarded by their properties of either small concentration
range or low sensitivity. Previous studies presented that the
1
threshold dose of O2 in PDT process was varied over a large range
from 0.1 mM to 12.1 mM.11 Thus, a fluorescent probe responding to
1O2 with a large concentration range is beneficial to monitor the
efficacy of PDT treatment. On the other hand, during the process of
PDT treatment, the scattered light may lead to producing small
1
dosage of O2 in the healthy cells adjacent to the cancer cells. The
elevated levels of 1O2 will induce severe damage to the healthy cells,
since the 1O2 can irreversibly oxidize various kinds of biological
molecules such as DNA, proteins, and lipids.12 Therefore, to prevent
the adverse effect of PDT treatment to healthy cells, fluorescent
probe should also display sufficient sensitivity to monitor small
dosage of 1O2 in the surrounding healthy cells. However, in general,
a fluorescent probe in response to analyte with large concentration
range is hard to respond to the analyte with high sensitivity,
or vice versa. Therefore, development of a fluorescent probe for 1O2
not only displaying large concentration range, but also showing high
sensitivity is a great challenge, but in urgent demand.
1
intricate process. The O2 concentration is drastically varied during
the PDT treatment, due to the PS distribution, photoirradiation
fluence rate, and oxygen concentration change dynamically and
interdependently in different individuals.4 Over the past few years,
understanding the biology of PDT has expanded, however, it remains
challenging to detect 1O2 concentration and track the related
dynamics, which is extremely crucial for achieving optimal efficacy
and safety of PDT treatment in clinic, especially when there is
curative intent. Consequently, construction of an efficient method
1
for detecting the O2 concentration during the PDT treatment is of
great significance.
Herein, we rationally developed an anthracene fluorophore
Among a variety of strategies for detection of 1O2, fluorescent
probe seems to be a superb tool for monitoring 1O2 in biological
system owing to their features of high sensitivity, simplicity, non-
invasiveness and potential for in vivo imaging.5 Currently, several
1
conjugated quinolinium, XQ-1, as a novel fluorescent probe for O2
1
(Scheme 1). Remarkably, probe XQ-1 was able to detect the O2 on
the basis of two-step cascade sensing reaction. The first step sensing
a. School of Chemistry and Chemical Engineering, Jiangsu University, Zhenjiang,
Jiangsu 212013 (P. R. China). E-mail: longlingliang@163.com,
wangkun@ujs.edu.cn
b. School of Medicine, Jiangsu University, Zhenjiang, Jiangsu 212013, (P. R. China).
E-mail: ahg5@ujs.edu.cn
c. Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science,
Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao
University of Science and Technology, Qingdao, Shandong 266042 (P. R. China).
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
Scheme 1 The sensing reaction of probe XQ-1 to 1O2 based on two-step
cascade sensing reaction, and the chemical structure of compound 3 and 4.
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reaction makes the probe detect O2 with high sensitivity, and the
second step sensing reaction renders the probe detect 1O2 with large
concentration range.
View Article Online
DOI: 10.1039/C9CC04300D
The synthesis and characterization of the probe XQ-1 were
displayed in supporting information (Scheme S1). The fluorescence
responses of probe XQ-1 to 1O2 were observed in blue fluorescence
channel and green fluorescence channel. In the absence of 1O2,
probe XQ-1 displayed almost no fluorescence (ФF = 0.004) in both
blue channel (Fig. 1a) and green channel (Fig. 1b). The quenched
fluorescence was probably attributed to the strong intramolecular
charge transfer (ICT) process from anthracene fluorophore to
quinolinium moiety.13 To illustrate these phenomena, time-
dependent density functional theory (TD-DFT) calculations were
carried out. The HOMO→LUMO transition (S0→S1) in probe XQ-1 was
allowable (f=0.4866), which showed that the electron on the
anthracene fluorophore was almost completely transferred to the
quinolinium moiety (Fig S1), implying the strong ICT. In addition, the
S1→S0 transition is forbidden (f=0.0000), which means that probe
XQ-1 is essentially nonfluorescent.
The responses of probe XQ-1 to 1O2 were investigated using
1
1
MoO42-/H2O2 system as the O2 source, as one O2 molecule can be
Fig. 1 Changes in fluorescence spectra of probe XQ-1 (20 μM) with 0-0.6 mM
1O2 in blue channel (λex = 368 nm) (a) and green channel (λex = 420 nm) (b);
Changes in fluorescence spectra of probe XQ-1 with 0.6-50 mM O2 in blue
quantitatively generated by reaction of two H2O2 under the catalysis
of MoO4 .
2- 14 When a small dosage of 1O2 (0-0.6 mM) was introduced,
1
the fluorescence of probe XQ-1 (20 μM) displayed dramatic channel (λex = 368 nm) (c) and green channel (λex = 420 nm) (d); Changes in
fluorescence intensity at 412 nm of probe XQ-1 with 0-0.6 mM 1O2 (e);
enhancement in the blue channel (ФF = 0.184) (Fig. 1a, 1e), and
Changes in fluorescence intensity at 506nm of probe XQ-1 with 0.6-50 mM
almost no fluorescence variations were noted in the green channel
1O2 (f). The error bars represent the standard deviation (n = 3).
(Fig. 1b). Thus, the sensing reaction was occurred after the probe
treated with a small dosage of 1O2. Moreover, the profiles of
1
was acquired when the probe was directly reacted with O2 in the
fluorescence emission spectra and excitation spectra in the blue
channel were resembled to that of anthracene 4 (Fig. S2, S3). These
results suggested that the anthracene fluorophore in the sensing
reaction product was intact. Furthermore, the results also inferred
period of first step reaction (Fig. S8). According to the ESI-Ms spectra,
together with the aforementioned fluorescence spectra and the
1
chemical reaction of O2,16 we tentatively proposed compound 1 as
the possible first step sensing reaction product (Scheme 1). In this
that the strong ICT process from anthracene to quinolinium moiety
product, quinolinium moiety was broken by an 1,4-cycloaddition
in the sensing reaction product was interrupted, which leads to the
reaction with 1O2, and thus the strong ICT process between
recovery of anthracene fluorescence. The tremendous fluorescence
anthracene moiety and quinolinium moiety was blocked, which
enhancement in blue channel upon treatment with a small dosage of
induced the fluorescence recovery of anthracene fluorophore. The
1O2 made probe XQ-1 sense 1O2 with high sensitivity. As shown in Fig.
second step sensing reaction product was successfully isolated, and
S4, the fluorescence intensity in the blue channel (I412) showed linear
1
the structure was identified to be compound 2 according to the H
relationship with the dosage of 1O2 in the range from 0 mM to 0.45
NMR spectroscopy (Fig. S9), ESI-Ms spectra (Fig. S10), fluorescence
emission spectra (Fig. S11), and fluorescence excitation spectra (Fig.
mM. And the detection limit was calculated to be 0.138 μM (S/N =
3).
S12). Based on the molecular structure of compound 2, a 1,2-
Interestingly, when more dosages of 1O2 were added (0.6 mM-50
cycloaddition reaction would be associated with the second step
mM), the enhanced fluorescence in the blue channel set out to
sensing reaction (Scheme 1). In addition, compared with the
attenuate (Fig. 1c). Meanwhile, an intense fluorescence in the green
channel centered at 506 nm was emerged and progressively
increased (ФF = 0.138) (Fig. 1d, 1f). These deduced that the first
fluorescence of compound 1 in the blue channel, the fluorescence of
compound 2 was red-shifted to the green channel. The red-shifted
fluorescence was attributed to weak ICT transition from anthracene
sensing reaction product further reacted with 1O2 to produce
moiety to the aldehyde moiety, which was illustrated by TD-DFT
another fluorescent product. The fluorescence intensity at 506 nm
calculation (Fig. S13). Therefore, the novel fluorescent probe could
showed good linear relationship with the logarithm of various dosage
1
detect O2 in two distinct fluorescence channels on the basis of the
of 1O2 in the range of 0.6 mM to 50 mM (Fig. S5). Thus, probe XQ-1
two-step cascade reaction. This makes the probe detect 1O2 with
also exhibited fluorescence response to 1O2 with very large
both high sensitivity and large concentration range.
concentration range, which is beneficial to monitor the efficacy
The UV-Vis absorption spectra responses of probe XQ-1 to 1O2
during the PDT treatment. In addition, probe XQ-1 displayed similar
were inspected. Probe XQ-1 itself exhibited two main absorption
peaks at 478 nm and 331 nm, respectively (Fig. S14). The broad
absorption peak at 478 nm is apparently ascribed to the transition of
ICT process from anthracene fluorophore to quinolinium moiety. The
absorption peak at 331 nm was due to the π-π* transition in the
quinolinium moiety. This can be illustrated by the TD-DFT calculation.
As shown in Fig S1, the HOMO-4→LUMO transition (f = 0.2266) is the
π-π* transition in the quinolinium moiety, which is responsible for
fluorescence response when the 1O2 was generated from
photoirradiation of PS TMPyP4 (5 μM) (Fig. S6-S7).
In view of the fluorescence response of probe XQ-1 to 1O2, we can
conclude that two-step cascade sensing reaction would be involved
in probe XQ-1 detecting 1O2. To further explore the sensing
mechanism, we tried to isolate and characterize the two sensing
reaction products. However, the first step sensing reaction product
was unstable in the process of isolation, which is frequently found in
the absorption peak at 331 nm. Moreover, by comparing the UV-Vis
absorption spectra of probe XQ-1 with that of N-ethyl-2-
1O2 related reaction products. 10a, 15 Fortunately, an ESI-Ms spectra
2 | J. Name., 2012, 00, 1-3
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methylquinolinium iodide 3 (Fig. S15), the absorption at 331 nm was
confirmed to be originated from the π-π* transition in the
quinolinium moiety. After treatment with small dosage of 1O2 (0-0.6
mM), the absorption peak at 478 nm decreased, denoting that the
strong ICT process from anthracene fluorophore to quinolinium
moiety was inhibited. Concomitantly, the absorption peak at 331 nm
was also decreased, inferring that the quinolinium structure was
broken, consistent with the structure of the proposed compound 1.
After reaction with 0.6 mM 1O2, several weak absorption peaks were
observed at 388 nm, 369 nm, 345 nm, and 326 nm, respectively.
These peaks are attributed to the π-π* transition of anthracene (Fig.
S16). When more dosage of 1O2 (0.6 mM-50 mM) was introduced, a
broad absorption peak at 409 nm was increased (Fig. S17), which was
ascribed to the transition of weak ICT process from the anthracene
to aldehyde in compound 2 (Fig. S13). Therefore, the variation of the
View Article Online
DOI: 10.1039/C9CC04300D
1
absorption spectra of probe XQ-1 to O2 further demonstrated the
two-step cascade sensing reaction.
To examine the detection specificity of probe XQ-1 to O2, XQ-1
1
Fig. 2 The fluorescence image of probe XQ-1 detecting endogenous 1O2 in
RAW 264.7 macrophage cells. (a)-(c): The fluorescence image in blue channel
of the cells stained with 2 μM XQ-1 in presence of 0 μM, 5 μM, and 20 μM
was treated with 0.6 mM various relevant species including reactive
oxygen species and metal ions. As displayed in Fig. S18, only 1O2 could
trigger obvious fluorescence enhancement at 412 nm, while other PMA, respectively; (d): The fluorescence image in blue channel of the cells
pre-treated with 100 μM His, and then stained with 2 μM XQ-1 in the
presence of 20 μM PMA; (e)-(h): The fluorescence imaging in green channel
of the cells corresponding to (a)-(d); (i)-(l): The bright field images of the cells
corresponding to (a)-(d); (m)-(n): Quantification of mean fluorescence
intensity in (a)-(d) and (e)-(h), respectively. The scale bar in (a)-(l) is 20 μm.
species did not induce any spectral changes. Furthermore, the
fluorescence color changes of XQ-1 to various species indicated that
1
XQ-1 was able to detect O2 by naked eyes. These results revealed
that the probe showed a highly selective response to 1O2. The
1
influence of pH value on the fluorescence response of XQ-1 to O2
was explored (Fig. S19). XQ-1 was capable of detecting 1O2 within the
pH range of 7-12, denoting that XQ-1 worked well at physiological
and alkaline pH condition. The effect of water volume fraction on XQ-
1 sensing 1O2 was also studied (Fig. S20). XQ-1 can be used for
detecting 1O2 even in the solution with 70% water. The photostability
blue channel and green channel (Fig. 3a,3d). Subsequently, the
selected cell region was continuously irradiated by 405 nm laser, and
the fluorescence imaging was acquired at different time interval.
After irradiation for 150 s, the cells in the irradiated region showed
intense fluorescence in the blue channel, while the cells in the
unirradiated region displayed no fluorescence (Fig. 3b, 3h). In
addition, no fluorescence was detected in the green channel (Fig. 3e,
1
of XQ-1 to O2 was further inspected. The results denoted that the
enhanced fluorescence in the blue channel and green channel was
stable under photoirradiation (Fig. S21-S22). In addition, the pseudo-
first-order rate constant (k′) was determined to be 0.04457 min-1 and
0.10379 min-1 for the probe reacted with 0.6 mM 1O2 and 50 mM 1O2,
respectively (Fig. S23-S24).
1
3i). These denoted that only small dosage of O2 was generated in
the irradiated cells region at the early stage of PDT process. Upon
continuous irradiation of the selected cells for 50 min, the
fluorescence in the blue channel of the irradiated cells was
significantly suppressed (Fig. 3c, 3h), whereas the fluorescence in the
green channel of the irradiated cells became pretty brighter (Fig. 3f,
To examine whether the probe is sufficiently sensitive to detect
1
small dosage of O2 in living system, probe XQ-1 was employed for
1
fluorescence imaging of endogenous 1O2 in RAW 264.7 macrophage
cells. After stained with 2 μM XQ-1 at 37oC for 30 min, the cells
exhibited negligible background fluorescence in blue channel (Fig. 2a)
and green channel (Fig. 2e). When XQ-1 stained cells were further
incubated with increasing dosage of phorbol myristate acetate (PMA,
3i), indicative of a large dosage of O2 being generated under the
continuous irradiation. Notably, the unirradiated cells surrounding
the irradiated cells region exhibited obvious fluorescence in the blue
channel (Fig. 3c, 3g, 3h), implying small dosage of 1O2 was generated
in these cells. Owing to the radial diffusion distance of 1O2 in an H2O-
incubated cells is only around 155 nm,19 the generated 1O2 was
unable to diffuse from the irradiated region to the surrounding
unirradiated region. Accordingly, the strong blue fluorescence in the
unirradiated cells was apparently ascribed to the scatter light
induced 1O2. The fluorescent probe itself has the possibility to
produce 1O2 under irradiation, which may interfere the 1O2
detection.10a This problem is frequently occurred in a commercially
1
an activator for endogenous O2 production),17 the fluorescence in
blue channel enhanced correspondingly (Fig. 2b-2c, 2m). Moreover,
in a control experiment, XQ-1 stained cells were pre-treated with
histidine (His, a 1O2 scavenger),18 and then incubated with PMA. No
visible fluorescence was observed in blue channel (Fig. 2d). Thus, the
fluorescence enhancement in the blue channel was indeed
originated from XQ-1 detecting endogenous 1O2. In addition, the
fluorescence in green channel displayed no evident change (Fig. 2e-
2h, 2n), denoting that only limited dosage of 1O2 was generated upon
the stimulation of PMA. These results established that probe XQ-1 is
sensitive enough to detect the small dosage of endogenous 1O2.
Next, probe XQ-1 was applied for imaging of intracellular 1O2
during the simulated PDT process. The 5-aminolevulinic acid (5-ALA)
induced protoporphyrin IX (PpIX) was used as PS.6f PC12 cells were
treated with 150 μg ml-1 5-ALA for 12 h, and then stained with XQ-1
for another 30 min. No background fluorescence was observed in the
1
available O2 probe Singlet Oxygen Sensor Green (SOSG).20 To rule
out this possibility, the PC12 cells were only incubated with XQ-1,
and then were continuously irradiated with 405 nm laser for 50 min.
No fluorescence was observed in the blue channel and green channel
(Fig. S25), signifying that probe XQ-1 itself cannot produce 1O2 under
irradiation. Therefore, during the PDT process, probe XQ-1 can be
used as a powerful tool to not only monitor 1O2 in the irradiated cell
region with a large concentration range, but also track the small
dosage of 1O2 in the unirradiated cell region with high sensitivity.
The colocalization experiments displayed that probe XQ-1 was
mainly localized in mitochondria (Fig. S26). This is presumably
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because the negative mitochondrial membrane potential (Δψ) allows
the positively charged probe to accumulate in mitochondria.21 The
cytotoxicity of probe XQ-1 to PC12 cells was evaluated by a standard
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay, and the results denoted that the cytotoxicity of the probe was
relative low (The cell viabilities remained above 90% after incubation
with 5 μM probe for 24 h) (Fig. S27).
Notes and references
View Article Online
DOI: 10.1039/C9CC04300D
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Fig. 3 Fluorescence imaging of 1O2 during the simulated PDT process. The
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In conclusion, we rationally constructed a novel fluorescent probe
for selectively detecting 1O2 on the basis of two-step cascade sensing
reaction. Notably, upon introducing small dosage of 1O2, the first step
sensing reaction was occurred, and the probe displayed pronounced
fluorescence enhancement in blue channel. As a result, the probe
could detect 1O2 with high sensitivity. However, upon further
1
introducing a large dosage of O2, the second step sensing reaction
was set out. The fluorescence of the probe in blue channel gradually
decreased, while the fluorescence in green channel emerged and
1
progressively increased. Therefore, the probe could also detect O2
with a large concentration range. The fluorescence imaging of
1
intracellular O2 during the simulated PDT process showed that the
probe was able to monitor 1O2 in the irradiated cell with a large
concentration range. Moreover, the probe was sensitive enough to
1
track the small dosage of O2 in the surrounding unirradiated cell.
Therefore, the probe will be employed as a versatile tool to not only
monitor the efficacy of PDT treatment to the cancer, but also can
track the adverse effect of the PDT treatment to the surrounding
healthy cells.
This research was supported by the National Natural Science
Foundation of China (21874059), and the Foundation of Key
Laboratory of Sensor Analysis of Tumor Marker, Ministry of
Education, Qingdao University of Science and Technology (No.
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DOI: 10.1039/C9CC04300D
1
A novel fluorescent probe for detecting O2 on the basis of two-step cascade reaction has been
rationally constructed.