A Turn-On Fluorescent Probe for Detection of Sub-ppm Levels of a Sulfur Mustard Simulant with High Selectivity

Article abstract of DOI:10.1021/acs.analchem.8b01057

A new type of fluorescent probe capable of detecting a sulfur mustard (SM) simultant at a concentration of 1.2 μM in solution and 0.5 ppm in the gas phase has been developed. Owing to its molecular structure with a thiocarbonyl component and two piperidyl

Full text of DOI:10.1021/acs.analchem.8b01057

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A Turn-on Fluorescent Probe for Detection of Sub-
ppm Level Sulfur Mustard Simulant with High Selectivity
Yuanlin Zhang, Yanlin Lv, Xuefei Wang, Aidong Peng,
Kaiquan Zhang, Xiaoke Jie, Jijun Huang, and Zhiyuan Tian
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01057 • Publication Date (Web): 12 Mar 2018
Downloaded from http://pubs.acs.org on March 12, 2018
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A Turnꢀon Fluorescent Probe for Detection of Subꢀ
ppm Level Sulfur Mustard Simulant with High
Selectivity
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Yuanlin Zhang a, Yanlin Lv a, Xuefei Wang a, Aidong Peng b, Kaiquan Zhang a, Xiaoke Jie a,
Jijun Huang b,* and Zhiyuan Tian a,*
a School of Chemical Sciences, University of Chinese Academy of Sciences, Beijing 100049, P.
R. China. Eꢀmail: zytian@ucas.ac.cn
b College of Materials Science and OptoꢀElectronic Technology, University of Chinese Academy
of Sciences, Beijing 100049, P. R. China. Eꢀmail: jjh@ucas.ac.cn
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ABSTRACT
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A new type of fluorescent probe capable of detecting sulfur mustard (SM) simultant with
concentration of 1.2 ꢀM in solution and 0.5 ppm in gas phase has been developed. Owing to its
molecular structure with thiocarbonyl component and two piperidyl moieties integrated into the
xanthene molecular skeleton, this probe underwent a highly selective nucleophilic reaction with
SM simultant and generated a thiopyronin derivative emitting intensive pink fluorescence. The
distinct difference in electronic structure between the probe and thiopyronin derivative generated
a marked shift of the absorbance band from 445 nm to 567 nm, which enabled an optimal
wavelength propitious to excite the thiopyronin derivative but adverse to the probe. Such
efficient separation of excitation wavelength and tremendous increase in fluorescence quantum
yield, from less than 0.002 to 0.53, upon conversion from the probe to thiopyronin derivative
jointly led to distinct contrast in the beaconing fluorescence signal (up to 850ꢀfold) and therefore
the unprecedented sensitivity for detecting SM species.
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Sulfur mustard (bis(2ꢀchloroethyl) sulfide, SM), a colorless to amber oily liquid with a
garlicꢀlike odor, is popularly known as mustard gas and was widely used as chemical war agent
in World War I and many military conflicts thereafter, leading to millions of casualties.1ꢀ3 Owing
to its high toxicity and powerful ability to incapacitate opponents, SM was called the “king of
war gases” and “chemical weapon of choice in modern tactical warfare”.4ꢀ6 Exposure to SM
typically leads to doseꢀdependent skin injury even areas of necrosis, eye injuries even permanent
blindness, respiratory failure even death.7,8 Additionally, SM may be carcinogenic and mutagenic
to human beings due to the chromosomal damage that it induced, especially for those undergoing
longꢀterm exposure.9 It is known that SM molecule generates toxicity based on the spontaneous
formation of a highly reactive and unstable sulfonium intermediate via an intramolecular
cyclization.10 Specifically, such cyclic intermediate is prone to react with a wide variety of
biological molecules carrying electronꢀrich moieties such as sulphydryl (ꢀSH) and amino (ꢀNH2)
groups of tissue macromolecules (e.g. proteins and nucleic acids), resulting in chromatid
aberrations and DNA strand breaks and apoptosis. Such acute damage, together with the rapid
inactivation of sulphydrylꢀcontaining proteins and peptides such as glutathione, is expected to
prevent cellular division and directly lead to programmed cell death. Alternatively, if cell death
is not immediate, the chromosomal damage may siginificantly increase the incidence of
malignancies for those suffered SM exposure.11, 12
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It is particularly noteworthy that victims after the initial exposure to SM typically undergo a
latent phase ranging from 30 min to 8 h before the development of toxic signs and symptoms.13
Such intoxication latent phase of SM typically delays decontamination efforts and therefore leads
to continued SM inhalation of victims suffered accidential exposure to SM. SM remains as a
serious public health and safety threat, especually for civilians and soldiers in conflict zones, in
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light of the virtual difficulty in verifying and controlling the manufacture or storage of SM and
its highly toxic feature. What is even more worse is that there have been no effective antidotes
for SM poisoning to date,14 which unequivocally highlights the vital significance of strategies for
promptly and reliably verifying trace amount of SM and the following emergency reponse.
According to the acute exposure guideline levels (AEGLs) established for guiding emergency
planning, prevention, and response in case of accidents or terrorist attacks with chemical agents,
SM with concentration at or below AEGLꢀ1 level, namely 0.06 ppm, does not cause observed
adverse effects on health for general public subject to exposure duration of 10 min.15,16
According to the Haber’s rule for guaging the toxic effect of poisonous gas, a dose schema of 10ꢀ
min exposure to 0.06 ppm poisonous gas is equivalent to acute exposure to 0.6 ppm gas with
duration time of 1 minute. Thus, the first and primary point of focus in terms of minimizing the
degree of SM injury shifts to reliable detection of trace amount of SM before its concentration
reaches to such safety ceiling level.
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A variety of strategies, including GC–MS,17ꢀ22 ion mobility spectrometry,23,24 quartz crystal
microbalance analysis,25 molecularly imprinted polymers,26 platinum (II) pincer,27 and
immunochemical methods28ꢀ30 have been reported for detection of SM. However, these strategies
generally suffer from disadvantages including poor portability, complicated operation, low
sensitivity and/or recognition selectivity. In contrast, fluorescenceꢀbased detection is highly
advantageous regarding sensitivity, spatiotemporal resolution, and ease of manipulation.31, 32
Based on a metalꢀion indicator displacement mechanism, Anslyn et al. developed the first SM
fluorescent probe with a detection limit of 0.2 mM for 2ꢀchloroethyl ethyl sulfide (2ꢀCEES),33 an
SM simulant that is chemically closely related to SM, known as “half mustard”, but free from the
associated toxicological properties.11,34 To take a step further, Anslyn et al. developed another
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type of probe for chromogenic and fluorometric detection of 2ꢀCEES with a detection limit of 10
ꢁM and the ability for gaseous SM sensing.35 Pardasani and coworkers reported a rhodamineꢀ
based fluorescent probe enables detection of gaseous SM at a level of 6.25 ppm/30 min with
naked eye.36 Despite significant advancement on SM sensing, there is still a pressing need of
fluorescent probes for promptly and reliably detecting SM with concentration below the AEGLꢀ1
safety ceiling level for minimizing the degree of SM injury. As consistent efforts in pursuit of
fluorescent probes for volatile hazardous chemicals sensing,37 we developed a pyroninꢀbased
fluorescent probe capable of detecting SM with concentration down to 0.6 ꢀM (in solution phase)
and presenting huge contrast in beaconing fluorescence signal upon exposure to 0.25 ppm
gaseous SM in this work. This is the first paradigm where a fluorescent probe enables facile and
highly selective detetcion of SM with concentration indeed below the AEGLꢀ1 safety ceiling
level (Table S1 in the Supporting Information), which providing an effective modality for
prompt detection of SM species in emergency events prior to the emergence of adverse effects
on health of general public exerted by such toxic substance.
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SM typically undergoes spontaneous first order intramolecular cyclization to form a
hyperactive episulphonium cation intermediate, which readily reacts with and alkylates
nucleophiles such as sulphydryl (ꢀSH) and amino (ꢀNH2) groups of proteins and acids.38
Benefiting from its large size and feature of easy polarization, sulfur atom is featured with easy
accessibility of its unshared electron pair and therefore very suitable to act as the counterpart
nucleophile.39 On the other hand, thiols are prone to undergo oxidation in the presence of oxygen
to form less ractive disulfide speices.38 With these in mind, we constructed a sulfurꢀbased
fluorescent probe, 3, 6ꢀdi(piperidinꢀ1ꢀyl)ꢀ9Hꢀxantheneꢀ9ꢀthione (DPXT), with thiocarbonyl
component and two electronꢀdonating piperidyl moieties integrated into the xanthene molecular
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skeleton in this work. Specifically, the thiocarbonyl component is expected to avoid the
undesirable SꢀS coupling of the probes while the 3, 6ꢀposition electronꢀdonating piperidyl
moieties augment the nucleophicility of sulfur, which greatly enhances the probe’s reactivity in
the SMꢀmediated nucleophilic reaction (Scheme 1).
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Scheme 1. Chemical structures of the fluorescent probe (DPXT) and analogues including
representative acylating agents, alkylating agent, sulphurꢀcontaining compounds and
hydrochloric acid investigated as interferent components and the proposed mechanism for 2ꢀ
CEES sensing.
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ꢀ EXPERIMENTAL SECTION
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Materials and Methods. N, Nꢀdimethylformamide (DMF), dimethyl sulphoxide (DMSO),
toluene, methanol, acetone, ethyl acetate (EA), acetonitrile, chloroform (CHCl3), 1, 2ꢀ
dichloroethane and dichloromethane (DCM) were purchased from Beijing Chemical Works
(Beijing, China), and dried before used. All deuterium solvents used in the nuclear magnetic
resonance spectroscopy (NMR) characterization were purchased from SigmaꢀAldrich. Silica gel
(200ꢀ300 mesh) for column chromatography was purchased from Qingdao Ocean Chemicals Inc.
All other chemicals were purchased from J & K Scientific, Ltd., and used without further
purification unless otherwise stated. NMR spectra were recorded in CDCl3, or DMSOꢀd6, or
CD2Cl2 at ambient temperature on a Bruker Avance 300 or JEOL JNMꢀLA400. Matrixꢀassisted
laser desorption ionizationꢀtime of flight (MALDIꢀTOF) mass spectra were recorded with a
Bruker BIFLEXꢀIII mass spectrometer. UV−vis absorption spectra were acquired on a UVꢀ2550
spectrophotometer (Shimadzu, Japan). Steadyꢀstate fluorescence spectra were recorded on
FluoroMaxꢀ4 spectrofluorometer (Horiba Jobin Yvon, NJ, USA).
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Determination of the Fluorescence Quantum Yield. The fluorescence quantum yield of
DPXT probe was estimated via the comparison of the integrated area of its corrected emission
spectrum with that of a perylene/EtOH solution as a standard (ϕ = 0.92).40 Likely, the
fluorescence quantum yield of thiopyronin derivative was estimated using the solution of
rhodamine B in EtOH solution as a standard (ϕ = 0.70).41
Determination of the Limit of Detection (LOD).The limit of detection was determined
based on the method reported in the previous literature.42 The fluorescence emission spectrum of
DPXT in DCM was acquired 10 times and the standard deviation of blank measurement was
achieved. Subsequently, a plot of the emission peak intensity (at 593 nm) against the
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concentration of 2ꢀCEES was obtained. The limit of detection was then determined based on the
3ꢀsigma method.
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Evaluation of Response of DPXT-loaded TLC Plate to Gaseous 2-CEES. A TLC plate
was patterned with “CEES” logo using a DPXT/DCM stock solution (100 µM) and then placed
in the air to evaporate the solvent. The photographs of TLC plate were firstly acquired under
illumination of indoor light and a UV lamp (Ex. 365 nm), respectively. 50 ꢁL of 2ꢀCEES/DCM
solution (1.1 mM) was added into a 2.5 L conical flask and then the lid was quickly closed.
Gently heating the conical flask generated a specific sealed space filled with 0.5 ppm gaseous 2ꢀ
CEES. The aforementioned TLC plate was put in such conical flask and then taken out 2 minutes
later for the counterpart photographs acquisition, respectively. Six parallel TLC plates were
loaded with DPXT by immersing one end of each plate in a DPXT/DCM stock solution (100
µM) and these plates were then placed in the air to evaporate the solvent. Subsequently, these
DPXTꢀloaded TLC plates after 1ꢀminute exposure to 2ꢀCEES gas with concentration of 0, 0.5,
1.0, 1.5, 2.0, 2.5 ppm, respectively, were imaged under illumination of 365ꢀnm light via the
identical procedure.
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Synthesis of 3, 6-Dihydroxy-9H-xanthen-9-one (2). 2, 2', 4, 4'ꢀTetrahydroxybenzoꢀ
phenone (Compound 1) (2.46 g, 10.0 mmol) in H2O (40 ml) was heated at 200 °C for 6 h in a
sealed tube. After cooling, the product was collected by filtration, washed with cold hexane and
dried, affording 2 as a white solid (2.28 g, 10.0 mmol, 100%). 1H NMR (400 MHz, d6ꢀDMSO): δ
6.81, (d, 2H, J = 2.2 Hz), 6.85 (dd, 2H, J = 8.8 Hz, 2.2 Hz), 7.98 (d, 2H, J = 8.8 Hz), 10.82 (s,
2H); 13C NMR (100 MHz, d6ꢀDMSO): δ 102.15, 113.69, 114.18, 127.89, 157.64, 163.37, 174.13.
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Scheme 2. Synthetic route to DPXT probe, thiopyronin derivative and reference compound
XTT.
Synthesis of 3, 6-Ditriflatexanthone (3). 3, 6ꢀDihydroxyxanthone (2) (2.28 g, 10.0 mmol)
was added to 300 ml DCM and pyridine (8.1 mL, 100 mmol). The resulting mixture was cooled
to at 0 °C. Then Tf2O (5.0 mL, 30.1 mmol) in CH2Cl2 was added dropwise. The reaction mixture
was warmed to room temperature and stirred for 24 hr. The reaction was quenched with H2O and
the organic layer was washed with H2O, 1 N HCl aq. and brine, and dried over Na2SO4. After
evaporation, the residue was purified by flash chromatography on silica gel (DCM/petroleum
ether =1 : 1) to give the target product (3.9 g, 7.9 mmol, 80%) as white needles. 1H NMR (400
MHz, CDCl3) δ 8.43 (d, 2H, J = 8.9 Hz), 7.47 (d, 2H, J = 2.3 Hz), 7.33 (dd, 2H, J = 8.9, 2.3 Hz);
13C NMR(100 MHz, CDCl3) δ 174.5, 156.6, 153.3, 129.6, 121.4, 118.7, 118.2, 111.4.
Synthesis of 3, 6-Di-piperidin-1-yl-xanthen-9-one (4). 3, 6ꢀDitriflatexanthone (3) (2.4 g,
5.0 mmol) was dissolved in 20 mL DMSO and piperidine (4.9 mL, 50.0 mmol) was added. The
reaction mixture was heated to 90 °C and stirred for 12 h. The solution was cooled to room
temperature and water was added. After treatment using water, the reaction mixture was poured
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into DCM (100ml) and the organic layer was washed with H2O, saturated Na2CO3 (aq.) and
brine, and dried over Na2SO4. After evaporation, the residue was purified by flash
chromatography on silica gel (DCM) to give the target product (1.6 g, 4.4 mmol, 90%) as yellow
solid. 1H NMR (300 MHz, d6ꢀDMSO) δ 7.86 (d, 2H, J = 9.0 Hz), 6.98 (dd, 2H, J = 9.0, 2.3 Hz),
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6.74 (d, 2H, J = 2.3 Hz), 3.41 (br, 8H), 1.60 (br, 12H); C NMR (75 MHz, d6ꢀDMSO) δ 173.0,
157.7, 154.8, 126.8, 111.7, 111.3, 98.8, 47.8, 24.8, 23.9. MALDIꢀMS: [M+H]+ calcd for
C23H26N2O2 362.1944; found 363.2068.
Synthesis of 3, 6-Di (piperidin-1-yl)-9H-xanthene-9-thione (5). 3, 6ꢀDiꢀpiperidinꢀ1ꢀylꢀ
xanthenꢀ9ꢀone (4) (0.34 g, 1.0 mmol) was dissolved in 20 mL toluene. Lawesson's Reagent (0.41
g, 1.0 mmol) was added. The reaction mixture was heated to 80 °C and stirred for 1 h. The
solution was cooled to room temperature and water was added. After treatment using water, the
reaction mixture was poured into ethyl acetate (50ml) and the organic layer was washed with
H2O and brine, and dried over Na2SO4. After evaporation, the residue was purified by flash
chromatography on silica gel (DCM) to give the target product (0.22 g, 0.58 mmol, 58%) as red
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solid. H NMR (CDCl3, 400 MHz): δ8.66(s, 1H), 8.64(s, 1H) 6.92(d, J =4.0 Hz, 1H), 6.89(d, J
=4.0 Hz, 1H), 6.61(s, 1H), 3.45 (m, 8H), 1.70 (m, 12H) ppm; 13C NMR (CDCl3, 100 MHz):
δ196.30, 155.3, 153.2, 131.7, 120.82, 98.21, 48.58, 25.4, 24.6 ppm; MALDIꢀMS: [M+H]+ calcd
for C11H12BF2N3H+: 378.1766, Found: 379.1879.
Synthesis of Thiopyronin (6). 3, 6ꢀdi (piperidinꢀ1ꢀyl)ꢀ9Hꢀxantheneꢀ9ꢀthione (5) (0.19 g,
0.5 mmol) was dissolved in 10 mL DCM. 2ꢀChloroethyl ethyl sulfide (5.8 mL, 50.0 mmol) was
added. The reaction mixture was stirred for overnight. After evaporation, the residue was
purified by flash chromatography on silica gel (DCM/MeOH=95:5) to give the target product
1
(0.20 g, 0.40 mmol, 80%) as red solid. H NMR (CDCl3, 400 MHz): δ8.26 (s, 1H), 8.23(s, 1H)
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7.23(s, 2H), 6.96 (s, 1H), 3.77 (m, 8H), 3.36 (t, 2H), 2.74 (t, 2H) , 1.79 (m, 12H), 1.19 (t, 3H)
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ppm; C NMR (DMSOꢀd6, 150 MHz): δ157.13, 156.70, 156.52, 131.73, 115.69, 97.13, 49.05,
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+
38.90, 31.91, 26.28, 25.36, 24.22, 15.21 ppm; MALDIꢀMS: [M]+ calcd for C11H12BF2N3 :
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467.2185, Found: 467.2186.
Synthesis of xanthene-9-thione (8). Xanthone (Compound 7) (0.20 g, 1.0 mmol) was
dissolved in 10 mL toluene. Lawesson's Reagent (0.41 g, 1.0 mmol) was added and then the
reaction mixture was heated to 80 °C and stirred for 1 h. The solution was cooled to room
temperature and water was added. After treatment using water, the reaction mixture was poured
into ethyl acetate (50ml) and the organic layer was washed with H2O and brine, and dried over
Na2SO4. After evaporation, the residue was purified by flash chromatography on silica gel
(petroleum ether/DCM=5:1) to give the target product (0.19 g, 0.90 mmol, 90%) as dark green
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solid. H NMR (400 MHz, CDCl3): δ8.76ꢀ8.73 (m, 2H), 7.78ꢀ7.74 (m, 2H), 7.51ꢀ7.49 (m, 2H),
7.40ꢀ7.35 (m, 2H); 13C NMR (100 MHz, CDCl3): 205.21, 150.55, 134.98, 129.93, 129.08,
124.83, 118.35; MALDIꢀMS: [M]+ calcd for C13H8OS+: 212.0, Found: 211.8.
ꢀ RESULTS AND DISCUSSION
Optical Response of DPXT to 2-CEES. As illustrated in Figure 1, DPXT probe in CH2Cl2
(DCM) solution clearly displayed UVꢀvis absorption and fluorescence emission spectra highly
dependent on 2ꢀCEES species. Specifically, the DPXT/DCM solution sample with pale yellow
color presented intense absorption feature in the range of 400ꢀ485 nm with maximum at ∼445
nm. Upon addition of small aliquots of 2ꢀCEES to the sample, a new absorption band in the
region of 480ꢀ640 nm with peak ∼ 576 nm and a shoulder at ∼530 nm, respectively, clearly
emerged. Additionally, such new absorption band was found gradually augment accompanying
with the concomitant decrease of the shortꢀwavelength absorption band upon further incremental
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addition of 2ꢀCEES. As a result, the color of the solution sample clearly changed from pale
yellow to orange. It is noted that such large bathochromic shift of absorption features of the
DPXTꢀcontaining sample upon reaction with 2ꢀCEES, from ∼445 to ∼576 nm, typically
generated a reaction product spectrally separated from the DPXT probe, which virtually enabled
optimal excitation wavelength for generating high fluorescence contrast in fluorescence sensing.
Additionally, DPXT probe displayed faint blue fluorescence upon excitation and presented a
quantum yield less than 0.002. In the presence of 2ꢀCEES, the highly nucleophilic sulphur atom
of thiocarbonyl moiety readily underwent nucleophilic reaction with the highly electrophilic
sulfonium ion intermediate; this reaction typically triggered ringꢀopening of the cyclic sulfonium
intermediate and transformation of probe into thiopyronin species with a fluorescence quantum
yield up to 0.53 because of the change in πꢀelectron delocalization features originating from the
transformation of thioketone to thioether form. The combination of huge separation in absorption
band and the appreciable increase in fluorescing ability of the probe upon reaction with 2ꢀCEES
is expected to generate remarkable beaconing fluorescence contrast for detetcing 2ꢀCEES with
high sensitivity.
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Figure 1. Evolution of UVꢀvis absorption (a) and fluorescence emission spectra (b) of
DPXT/DCM solution upon gradual addition of 2ꢀCEES. (c) A plot of the fluorescence peak
intensity (at 593 nm) against the concentration of 2ꢀCEES. The red line is the linear calibration
curve with regression coefficient (R2) of 0.994. (d) Photographs of DPXT/DCM solution before
and after addition of 100 equiv. of 2ꢀCEES under room light (top) and UV light (down)
illumination. [DPXT] = 10 ꢀM.
The excitation wavelength for acquiring fluorescence spectra of the DPXT/DCM sample in
the presence of 2ꢀCEES (Figure 1b) was 570ꢀnm. It can be clearly seen from Figure 1a that such
excitation wavelength is not within absorption region of DPXT but very close to the optimum
excitation wavelength of the reaction product (~576 nm). Thus, large beaconing signal contrast
and high sensitivity to 2ꢀCEES of the probe are expected in fluorescence sensing. Upon
excitation at 570 nm, the DPXT/DCM sample without 2ꢀCEES hardly fluoresced, as shown in
Figure 1b. In sharp contrast, a new emission band in the range of 580−720 nm with peak at ∼
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593 nm clearly emerged and gradually augmented upon incremental addition of 2ꢀCEES,
eventually generated a solution sample emitting vivid pink fluorescence. Specifically, addition of
∼100 equiv. of 2ꢀCEES markedly generated enhancement of the beaconing fluorescence signal
(at 593 nm) more than 850ꢀfold. Figure 1c displays the fluorescence titration result of the DPXT
probe sample with the aliquots of 2ꢀCEES ranging from 0 to 600 ꢁM. To verify response feature
of DPXT probe to 2ꢀCEES with lower concentration range, fluorescence response of DPXT
probe to 2ꢀCEES with concentration in range of 0ꢀ5ꢁM was investigated. Specifically, perfect
linear relationship between the fluorescence signal intensity and the concentration of 2ꢀCEES
was obtained (Figure S3), which presented a detection limit of 1.2 ꢀM for 2ꢀCEES sensing. Such
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sensitivity is equivalent to ∼0.6 ꢀM of real SM agent in light of the “half mustard” nature of 2ꢀ
CEES and about 10 times superior than the counterpart SM sensing ability reported
previously,30,33,35,36,43 indicating the superiority in detection sensitivity of DPXT probe. We
noticed that an acridinethioneꢀbased SM probe with similar sensing mechanism and a limit of
fluorescence detection of ∼31.6 ꢀM (0.005mg/mL) was reported very recently,44 which is more
than 52 times coarser than the detection sensitivity that DPXT probe enabled ( 0.6 ꢀM). Such
superior sensitivity that DPXT probe displayed can be attributed to the sharp contrast between
the intrinsic electronic structure features of DPXT molecule and the SMꢀmediated reaction
product (thiopyronin derivative), as discussed in the next section.
Recognition Specificity of DPXT to 2-CEES. To gauge the recognition specificity of
DPXT probe to 2ꢀCEES, the fluorescence responses of DPXT/DCM sample to interferent
substances with similar reactivities such as acylating agents, alkylating agent, sulphurꢀcontaining
compounds, and hydrochloric acid were investigated. As illustrated in Figure 2a, fluorescence
emission features of the DPXT probe sample nearly remained unchanged in the presence of
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various interferent substances as compared to the free DPXT probe sample even if the
concentration of these interferent substances largely exceeded that of the probe (200 equiv.). In
marked contrast to such almost unresponsive feature, 2ꢀCEES with half concentration of the
interferent substances (100 equiv.) clearly induced more than 850ꢀfold augmentation of the
beaconing fluorescence signal, unequivocally indicating a high recognition specificity of DPXT
for 2ꢀCEES sensing over similarly reactive toxic reagents. As illustrated in Figure 2c,d, DPXTꢀ
containing solution samples in the presence of 2ꢀCEES and interfering substances, respectively,
clearly presented distinct contrast in both vision and fluorescence mode, suggesting a facile way
of monitoring SM using DPXT probe.
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Figure 2. (a) Fluorescence emission spectra of the DPXT/DCM sample (10 ꢁM) with and
without of 2ꢀCEES (100 equiv.) and interferent substances (200 equiv.), respectively. (b)
Fluorescence peak intensity (at 593nm) of the DPXT/DCM sample in the presence of 2ꢀCEES
and various interferent substances. Photographs of DPXT (10 ꢁM) in the absence and presence
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of 2ꢀCEES, part typical interferent substances (c) under indoor light and (d) the illumination of a
handꢀheld UV lamp (Ex. 365 nm).
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Response of DPXT-loaded Substrate to Gaseous 2-CEES. SM is hazardous to humans in
both liquid and vapor form.26 Thus, one of the key litmus test for SM probes is whether they are
capable of detecting SM vapor with concentration at or below the margin of safety (AEGLꢀ1
level), especially for practical detection applications in which warm climates facilitate
vaporization of SM26. To evaluate the effectiveness of DPXT probe for detecting SM in gas
phase, the fluorescence response of DPXTꢀloaded substrates to gaseous 2ꢀCEES was
investigated. In a typical protocol, a DPXTꢀpatterned TLC plate with logo of “CEES” was
imaged before and after exposure to 0.5 ppm 2ꢀCEES vapor, respectively. It was found that the
DPXTꢀloaded TLC plates after treatment in vapor presented marked contrast in color and
fluorescence brightness to those of the plate prior to 2ꢀCEES vapor treatment (Figure 3a).
Specifically, the logo of “CEES” on the patterned TLC plate before exposure to 2ꢀCEES vapor
was hardly distinguishable from the DPXTꢀfree part upon illumination of UV light. In sharp
contrast, the patterned TLC plate after exposure to 2ꢀCEES vapor clearly presented the logo of
“CEES” with very clear edge from the DPXTꢀfree part, fluorecing vivid pink color upon
illumination of UV light. Additionally, such DPXTꢀloaded TLC plate after exposure to 2ꢀCEES
vapor presented pink fluorescence ∼16 times brighter than that of the plate before exposure to 2ꢀ
CEES vapor (Supporting Information), indicating the high sensitivity of such DPXTꢀloaded
platform for gaseous SM sensing.
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Figure 3. (a) Photographs of a TLC plate patterned with “CEES” using DPXT/DCM solution
before and after 1ꢀmin exposure to 0.5 ppm 2ꢀCEES vapor acquired via illumination of indoor
light and UV lamp, respectively. (b) Photographs of six parallel TLC plates with one end of each
loaded with DPXT after exposure to 2ꢀCEES vapor with concentration 0f 0, 0.5, 1.0 1.5, 2.0, 2.5
ppm acquired via illumination of UV light.
To evaluate the sensing limit of DPXT to gaseous SM, six parallel DPXTꢀloaded TLC
plates were prepared via the same procedure and their photographs before and after exposure to
gaseous 2ꢀCEES with various concentration were acquired under illumination of 365ꢀnm UV
light. Specifically, the concentration of 2ꢀCEES gas in six sealed containers was 0, 0.5, 1.0, 1.5,
2.0, 2.5 ppm, respectively, and the exposure duration was 1 minute in all cases. The TLC plate
underwent exposure to atmosphere without 2ꢀCEES gas (0 ppm gas) did not display noticeable
boundary line between its DPXTꢀloaded end and the DPXTꢀfree end, both presenting vivid blue
color throughout the whole TLC plate (Figure 3b). In sharp contrast, the DPXTꢀloaded end of
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TLC plate after exposure to 0.5 ppm 2ꢀCEES gas clearly presented pink fluorescence upon
illumination of UV light and therefore generated a clear boundary line between the DPXTꢀ
loaded end and the DPXTꢀfree end. Upon increasing the concentration of 2ꢀCEES gas that the
TLC plates were exposed to, color and brightness contrast between the DPXTꢀloaded end and
the DPXTꢀfree end obviously increased, which is in line with the fluorescence analysis results in
the case of solution DPXT probe sample for 2ꢀCEES sensing in its liquid form (Figure 1c).
These results indicate that the platform with DPXT immobilized on the TLC plate is capable of
reporting the exixtence of 2ꢀCEES gas with concentration down to 0.5 ppm in the case of acute
exposure with duration of 1 minute.
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It is noted that the lower limit concentration of 2ꢀCEES vapor in the aboveꢀmentioned 2ꢀ
CEES gas sensing experiments was 0.5 ppm, which is equivalent to 0.25 ppm of gaseous SM11, 34
Such detection sensitivity to SM, namely 0.25 ppm/min, is much higher than the highest
sensitivity regarding SM fluorescence detection reported to date, namely 6.25 ppm/30 min.36
Wolfbeis et al recently reported enzymeꢀbased platform enabling photographic detection of SM
gas with dose schema level down to 4.2 ppm/min (3 ꢁg/L on the basis of 10ꢀmin exposure
duration),30 which is a significant progress regarding SM sensing. According to the AEGLꢀ1
safety criterion regarding SM exposure, namely acute exposure at 0.6 ppm/min level, the
detection sensitivities of all these probes/sensors reported to date are not sufficient for providing
alert signals when the concentration of SM reaches the ceiling level. In marked contrast, the
detetcion limit that our DPXT probe enabled (0.25 ppm/min) is indeed below such safety ceiling
level, to which individual may be exposed in accidental cases of acute exposure to SM.15,16
Unequivocally, DPXT probe developed in this work displayed sensing ability for providing
timely alert signals before accumulation of SM exerts adverse effects on human health.
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Sensing Mechanism. The high sensitivity that DPXT probe exhibited is determined by its
unique molecular structure. As discussed above, using sulfur as active site enabled the high
reactivity of DPXT probe toward SM species by maximizing it nucleophicility. Moreover, the
intrinsic electronic structure feature of thiocarbonylꢀcontaining DPXT molecule endowed limited
πꢀconjugation and therefore highꢀenergy absorption to the probe. On the other hand, SMꢀ
mediated nucleophilic reaction of the DPXT probe yielded the thiopyronin derivative with two
nitrogen atoms of the piperidyl substituents involved in delocalization of the positive charge
from the core heterocyclic framework, which is characterized with absorption and emission
features at significantly lower energy regions as compared to other counterpart pyronin
derivatives.45 To shed light on the key role of the piperidyl substituents, we synthesized a
reference compound with similar molecular skeleton but without the piperidyl substituents,
xantheneꢀ9ꢀthione (XTT), and investigated its optical responses to 2ꢀCEES (Figure S5). In sharp
contrast to DPXT, the reference compound XTT after exposure to 2ꢀCEES did not display
appreciable absorption in the range of 480ꢀ640 nm and therefore can not be excited by 570ꢀnm
light. As a result, no appreciable fluorescence enhancement of XTT upon exposure to 2ꢀCEES
was observed. Such performance of this reference compound definitely verified the key role of 3,
6ꢀposition electronꢀdonating piperidyl moieties in DPXT for enabling the fluorescence response
features. The resultant marked difference in the maxima of absorption band, up to 131 nm,
virtually enables sufficient spectral separation of the thiopyronin dye from the DPXT probe and
therefore optimal excitation wavelength for generating high fluorescence contrast for SM sensing.
Additionally, the thiopyronin derivative is generally featured with much higher fluorescence
quantum yield (ϕ = 0.53) as compared to that of the DPXT probe (ϕ less than 0.002), which
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provides another enabling factor contributing to high fluorescence contrast for SM sensing with
high sensitivity.
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To conclude, we have developed pyroninꢀbased fluorescent probe, DPXT, capable of
detecting SM with limit of detection down to 0.6 ꢀM in solution phase and 0.25 ppm/min SM in
gas phase, respectively, which is the first fluorescent probe enabled detecting gaseous SM with
concentration indeed below the AEGLꢀ1 level (0.6 ppm/min). Such nearly nonfluorescent DPXT
probe readily react with SM via nucleophilic reaction process and therefore yielded highly
fluorescent thiopyronin derivative, which yielded distinct contrast in fluorescence emission
features between the reaction product and the probe. The excellent recognition specificity of the
DPXT probe to SM species was also verified. The proposed sensing mechanism was sufficiently
supported by UVꢀvis absorption and fluorescence emission spectroscopy, NMR, mass spectrum
characterization results, as well as the response features of reference compound (Supporting
Information). Such unprecedented detection sensitivity and excellent recognition specificity that
DPXT probe displayed unequivocally proclaimed its potential for prompt and reliable detection
of highly hazardous SM species characterized with latent phase of intoxication in practical
applications.
ꢀ ACKNOWLEDGMENTS
This work was financially supported by the National Natural Science Foundation of China (grant
no. 21373218 and 21573234) and the Instrument Developing Project of the Chinese Academy of
Sciences (Grant YZ201455).
Supporting Information.
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Reaction time studies, screening of solvent, partial H NMR (400 MHz) spectral changes of
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DPXT probe in CD2Cl2 in the absence and presence of 2ꢀCEES, selected comparison of
fluorescent probes for 2ꢀCEES or sulfurs mustard as well as copies of NMR spectra and
MALDIꢀMS.
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AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: zytian@ucas.ac.cn; jjh@ucas.ac.cn
Notes
The authors declare no competing financial interest.
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