A novel fluorescence probe based on: P -acid-Br and its application in thiourea detection

Article abstract of DOI:10.1039/c6ra06953c

In this paper, a novel phenyleneethynylene derivative 4,4′-(2,5-dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl) dibenzoic acid (p-acid) and its derivative p-acid-Br were synthesized. Infrared spectroscopy (IR), fluorescence (FL) spectroscopy and ultraviolet

Full text of DOI:10.1039/c6ra06953c

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A Novel Fluorescence Probe Based on P-acid-Br and its
Application in Thiourea Detection
Cite this: DOI: 10.1039/x0xx00000x
a
a
, a
b
a
Xiu Wang , Chunlei Yang , Mei Yan* , Shenguang Ge , Jinghua Yu
In this paper,
a
novel phenyleneethynylene derivative 4,4’ꢀ(2,5ꢀdimethoxyꢀ1,4ꢀ
Received 00th January 2012,
Accepted 00th January 2012
phenylene)bis(ethyneꢀ2,1ꢀdiyl) dibenzoic acid (pꢀacid) and its derivative pꢀacidꢀBr were
synthesized. Infrared spectroscopy (IR), fluorescence (FL) spectroscopy and ultraviolet visible
(UVꢀvis) spectroscopy were applied to characterize pꢀacid and pꢀacidꢀBr. To research the
practical applicability, a sample thiourea sensor was constructed using pꢀacidꢀBr label, in
which the FL intensity response was proportion to the thiourea concentration in the range of
DOI: 10.1039/x0xx00000x
www.rsc.org/
0
.5~1000 nM, with a detection limit of 0.26 nM. Furthermore, the sensor showed high
specificity, excellent stability, and good reproducibility. The pꢀacidꢀBr ꢀbased thiourea sensor
can also provide potential application for detection of other organics. The method showed low
detection limit, good specificity, high sensitivity and reproducibility. Satisfactory results were
obtained for determination of thiourea in various water samples and fruit juice samples. This
work is to open new avenues in the application of phenyleneethynylene derivative for sensitive
thiourea assay. Hence, the proposed fluorescence sensor could become a promising method for
local market.
1
6
carbohydrates and result in carcinogenic for humans. For these
reasons, thiourea and its derivatives are considered as toxic and
hazardous are forbidden to appear in the environment. Therefore, it
is necessary to develop a determination method for thiourea and its
derivatives in real samples, such as water, soil, or fruits. Several
methods have been employed in the determination of thiourea:
Introduction
Thiourea, a sulfurꢀcontaining organic compound, has many
industrial applications. Thiourea and its derivatives have been
widely applied in various fields, including industry, medicine,
1
ꢀ4
agriculture and chemistry. The reaction of thiourea with hydrogen
peroxide under certain conditions produces a powerful reductive
bleaching agent which is routinely used in the textile industry.
Thiourea and its derivatives are known corrosion inhibitors. For
instance, thiourea is usually used for electrodeposition of metals,
rubber vulcanization, cleaning, detection, and leaching of gold in
1
7
18
raman spectroscopy,
mass spectrometry,
fourier transform
infrared spectroscopy, high performance liquid chromatography
and fluorescence.
1
9
20
Fluorescence based detection, homogeneous systems that allow
direct measurement of binding in solution, has played significant
role at the forefront of the bioꢀanalytical area, because it possesses
21
5ꢀ8
industry.
In agriculture, thiourea and its derivatives are also
employed as a fertilizer, fungicide for some fruits, and inhibiter for
the dormancy of seeds and tubers.
appearing in industrial waste water, river water, on the surface of
fruits, and even in fruit juices. The boiler chemical cleaning wastes
are then disposed of according to the guidelines established by
22,23
9
,10
ultrahigh sensitivity and selectivity.
One of the most exciting
As a result, thiourea might be
aspects of fluorescence technology is that it can decrease the size of
a sample down to the singleꢀmolecule detection level, and it is this
fact that provides an opportunity for miniaturization and high
throughput screening. In addition, fluorescence has enabled the
elucidation of many biological processes, studying the structure and
dynamics of biological macromolecules and determining the trace
amounts of biological macromolecules. Fluorescence sensors
employ a fluorescent signal for the detection of analysis. In order to
detect different organics at an earlier stage of a disease development,
a low detection limit of the organics is desirable.
2
4
1
1
environmental agencies. Due to its significant role in many fields,
thiourea is confirmed as a typical contaminator and prohibited in the
environment due to its serious toxicity to the environment and
25
12
human health. It is also used as reagent for spectrophotometric
determination of metals. Thiourea is less toxic than cyanide but it is
still a toxic substance, dangerous for its effects on carbohydrate
metabolism, carcinogenic, potentially allergenic, showing in addition
13ꢀ15
inhibitory effects on nitrification in soils and water.
High
Among fluorescenceꢀbased detection, conjugated compounds
are stars of fluorescent dyes for their wellꢀknown high absorption
coefficients and high fluorescence efficiency, which lead to a wide
concentrations of thiourea in industrial wastes may not be acceptable
because of its high oxygen demand and organic nitrogen content. For
example, thiourea could cause allergy, hypothyroidism, and other
glandular diseases. Organic sulfur compounds are common in anoxic
environments of wastewater and sediments and are environmentally
significant due to their offensive odor. Besides, thiourea is toxic and
a cancer support agent, from the above mentioned hazards, thiourea
and its derivatives could also disturb the metabolism of
2
6ꢀ31
range of applications in optoelectronic sensors.
Phenylene
ethynylenes are an interesting kind of rigid rod molecular system
which maintains pꢀelectron conjugation at any degree of rotation of
the phenyl rings and is proposed as a linker for electronic
3
2,33
communication in molecular electronic devices.
Phenylene
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DOI: 10.1039
J
/
o
C
u
6
r
R
n
A0
a
6
l
9
N
5
a3Cme
ethynylenes possess tunable optical properties both in solution and in
PꢀacidꢀBr was synthesized according to a previous protocol
3
7
the solid state. They are proposed as components in optoelectronic with major modifications , the procedure is shown in Scheme 1.
sensors. For sensor applications, it is extremely important to
34,35
understand the selfꢀorganization of phenylene ethynylenes,
since
Synthesis of 2 : To a solution of 1,4ꢀdimethoxy benzene (2.76
the interchromophoric interactions significantly influence their g, 20 mmol) in 20 mL of acetic acid, 2 mL of water and 1 mL of
36
sensor properties. In an effort to prepare an efficient and uniform concentrated H SO , KIO (5.52 g, 24 mmol) and I (6.09 g, 24
2 4 4 2
fluorescence probe, the phenylene ethynylene derivative possessing mmol) were added. The reaction mixture was stirred at 120 °C for 24
carboxylic acid groups at the paraꢀposition was called pꢀacid and its h and then cooled to room temperature. The excess iodine was
ramification pꢀacidꢀBr which make it reasonable to predict that they removed by the addition of sodium thiosulphate solution until the
may have potential capability in biological and organic brown color of iodine disappeared. The excess
macromolecules analysis.
neutralized with Na CO solution until the brisk effervescence
stopped. The residue was extracted with chloroform. The white solid
H SO was
2 4
2
3
In this study, we report a novel fluorescence probe for targeted product (7.79 g, 69%) was separated using a silica column with
imaging FL overexpressed cancer cells based on the selfꢀassembly of hexane as eluent.
thiourea and pꢀacidꢀBr. The primary fluorescence of pꢀacidꢀBr turns
on first upon the electrostatic adsorption of thiourea onto pꢀacidꢀBr
based on cyclization reaction to produce a probe with negligible tetrahydrofuran (THF) (1:3), compound 2 (1.17 g, 3.90 mmol), CuI
fluorescent background. The competition of thiourea expressed in (0.023 g, 0.12 mmol) and Pd(PPh Cl (0.020 g, 0.03 mmol) in
Synthesis of 3: To the solvent of triethylamine and
)
3
2
2
the cancer cells enables thiourea desorption from pꢀacidꢀBr. Thus, 37.72 mL of diisopropylamine, trimethylsilylacetylene (TMSA)
the electron transfer between thiourea and pꢀacidꢀBr is inhibited. In (0.90 mL, 6.40 mmol) was slowly added. The mixture was stirred at
this way, thiourea overexpressed cells are illuminated. We first reflux using iceꢀcold condensing circulation for about 3 h. The
examined the effect of the solvent polarity on the fluorescence of pꢀ reaction mixture passed through a short celite column and further
acid and then explored thiourea in water and selective detection of purified by column chromatography using silica column with
thiourea in cells with pꢀacidꢀBr. The identification and quantification petroleum ether/methylene chloride (4:1) as eluent to obtain white
of thiourea in living cells are significant issues in medical and crystals of compound 3 (0.93 g, 72%).
clinical research, given that thiourea has severe toxic effects and
have been implicated in hypothyroidism. For these reasons,
establishing a facile, rapid, highly selective and sensitive method for methanol (25 mL) and K
Synthesis of 4: To a solution of 3 (0.58 g) in THF (25 mL),
CO (0.974 g, 7.06 mmol) were added and
2
3
the detection of thiourea is a great interest.
the mixture stirred for 5 h. The solvent was evaporated and the
residue was poured into water and extracted with chloroform. The
organic layer was washed with saltꢀsaturated solution and dried over
anhydrous sodium sulphate. A yellow solid product (0.27 g) was
obtained after the solvent was removed.
Experimental
Reagents
Synthesis of 5: To a solution of 4 (1.16 g, 6.24 mmol), CuI
0.118 g, 0.62 mmol), methyl 4ꢀiodobenzoate (3.27 g, 12.48 mmol)
All reagents were of analyticalꢀreagent grade or the highest
purity available. Dimethylsulfoxide (DMSO), sodium hydroxide
(
and Pd(PPh ) Cl (0.2 g, 0.29 mmol) in triethylamine and THF (1:3)
(
(
(
(
NaOH), potassium hydroxide (KOH), paraꢀdimethoxybenzene
3 2
2
was added and refluxed for 12 h. The residue was concentrated and
the yellow solid product (2.21 g) purified by column
chromatography over silica using methylene chloride/petroleum
ether (2:1) as eluent.
C H O ), N,N’ꢀdimethylformamide (DMF), sodium sulfite
8
10
2
Na SO ), tetrahydrofuran (THF), methanol (CH OH), ethanol
2
3
3
CH CH OH), aqueous ammonia (NH OH, 25%) and TEOS were
3
2
4
obtained from Sinopharm Chemical Reagent Co., Ltd (Shanghai,
China). Bis(triphenylphosphine)ꢀ palladiumchloride (Pd(PPh ) Cl ),
3
2
2
Synthesis of 6: To a solution of HPLC purified 5 (0.1 g) in THF
15 mL), KOH (1.0 g) and methanol (8 mL) were added and refluxed
iodine (I2), cuprous iodide (CuI), triethylamine ((C H ) N),
2
5 3
(
potassium iodate (KIO ) and trimethylsilylacetylene (TMSA) were
3
at a temperature of 70 ° C for about 5 h. The reaction mixture
neutralized with dilute HCl to remove excess alkali and 50 mL water
was added to precipitate the product. The mixture extracted with
chloroform and the combined chloroform layer was evaporated
under reduced pressure to yield the yellow solid product 6 (0.09 g).
purchased from Alfa Aesar China Ltd. Acetic acid (CH COOH),
3
sulphuric acid (H SO , 98%), hydrochloric acid (HCl, 37.5%),
2
4
sodium carbonate (Na CO ), ammonium chloride (NH Cl), sodium
2
3
4
chloride (NaCl), methylene chloride (CH Cl ), chloroform (CHCl ),
2
2
3
ethoxyethane (C H OC H ), magnesium sulfate (MgSO ), sodium
2
5
2
5
4
sulfate (Na SO ) and petroleum ether (30ꢀ60°C) were obtained from
2
4
ꢀ
1
Synthesis of 7: To a solution of 6 (0.58 g) in THF (25 mL),
methanol (25 mL) and CuI (1.724 g) were added and the mixture
stirred for 12 h. The solvent was evaporated and the residue was
poured into water and extracted with chloroform. The organic layer
was washed with saltꢀsaturated solution and dried over anhydrous
sodium sulphate. A light brown solid product pꢀacidꢀBr (0.27 g) was
obtained after the solvent was removed. The product was used to
detect the concentration of thiourea.
SigmaꢀAldrich. The 0.1 mol⋅L phosphate buffer solution (PBS),
which was made from Na HPO , NaH PO , and H PO , was
2
4
2
4
3
4
employed as a supporting electrolyte.
Apparatus
Infrared (IR) spectra were recorded using a Nicolet 400 Fourier
transform infrared spectrometer (Madison, WI). Absorption spectra
were recorded using a UV3101 spectrophotometer. Fluorescence
excitation and emission spectra were obtained with a PerkinꢀElmer
LSꢀ55 luminescence spectrometer.
Fluorescence intensity detection
For the detection of fluorescence intensity, thiourea of various
concentrations was added into the solution of pꢀacidꢀBr (dissolved in
Synthesis of p-acid-Br
ꢀ
1
PBS containing 1 mol⋅L DMF, pH7.4) and incubated for 60 min at
2
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DOI: 10.1039/C6RA069 C
room temperature. Subsequently, the fluorescence intensity of
thiourea was detected.
To further monitor the formation of pꢀacid, pꢀacidꢀBr and pꢀ
acidꢀBrꢀthiourea, the UVꢀvis absorption spectrum (Figure 1C) of pꢀ
acid (curve a), pꢀacidꢀBr (curve b) and pꢀacidꢀBrꢀthiourea (curve c)
were also investigated. The pꢀacid (curve a on Figure 3) showed two
absorption peaks at 382 and 456 nm, while there was one peak at
2
81 nm for the pꢀacidꢀBr (curve b on Figure 1C). When pꢀacidꢀBrꢀ
thiourea was prepared (curve c on Figure 1C), the peak at 443 nm
were mainly ascribed to the molecule π ꢀconjugated structure.
ꢀ
1
Figure 1 (A) The fluorogram of the 50 mmol⋅L pꢀacid (curve
a for Ex, curve b for Em). (B) Emission spectra recorded from (a) pꢀ
acid, (b) pꢀacidꢀBr and (c) pꢀacidꢀBrꢀthiourea, (the excitation
ꢀ
1
wavelength is 396 nm, slit: 5 nm/5 nm) in 10 mmol⋅L PBS at pH
ꢀ
1
7
.4 containing 1 mol⋅L DMF. (C) UVꢀvis spectra recorded from (a)
pꢀacid, (b) pꢀacidꢀBr and (c) pꢀacidꢀBrꢀthiourea solution in 10
ꢀ
1
ꢀ1
mmol⋅L PBS at pH 7.4 containing 1 mol⋅L DMF. (D) Infrared
spectra of compounds: (a) pꢀacid, (b) pꢀacidꢀBr and (c) pꢀacidꢀBrꢀ
thiourea.
Scheme 1 Synthesis procedure of pꢀacidꢀBr and detect of
thiourea.
Result and discussion
FT-IR analysis and NMR analysis
Absorption and emission
FTꢀIR analysis (Figure 1D) was performed to characterize pꢀ
acid (curve a), pꢀacidꢀBr (curve b) and pꢀacidꢀBrꢀthiourea (curve c).
The prepared pꢀacid exhibited a blue color under ultraviolet
radiation (λ = 365 nm). The fluorescence spectrum of pꢀacid is
shown in Figure 1A: excitation into the long absorption band
produces roomꢀtemperature photoluminescence and the maximum
fluorescent emission was obtained at the wavelength of 481 nm,
indicated that the pꢀacid molecule has favorable properties for
fluorescence. Moreover, the small energy difference between
excitation and emission spectra indicates the existence of a πꢀ
conjugated structure. Moreover, as Figure 1B showed the FL
spectrum of pꢀacid (curve a), pꢀacidꢀBr (curve b) and pꢀacidꢀBrꢀ
thiourea (curve c) were also investigated. We observe that the pꢀacid
have the excitation wavelength (Ex) at 376 nm and emission
wavelength (Em) at 438 nm. The pꢀacidꢀBr do not have a FL
intensity, because the C=O and Brꢀ have quenching the FL intensity
of pꢀacid. The Em positions between the pꢀacid and pꢀacidꢀBrꢀ
thiourea have a clear distinction, which shows that there have
chemical bonds between the pꢀacidꢀBr and thiourea. Nevertheless,
the FL intensity of pꢀacidꢀBrꢀthiourea is greatly increased compared
to pꢀacid.
ꢀ1
ꢀ1
ꢀ1
The strong peaks at 1644 cm , 1498 cm and 1484 cm (curve a);
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ1
1
657 cm and 1546 cm 1398 cm (curve b); 1602 cm , 1508 cm
ꢀ1
and 1492 cm (curve c) belong to C=C stretching vibration modes of
ꢀ1
ꢀ1
ꢀ1
benzene. The peaks at 2959 cm , 2930 cm and 2832 cm (curve a);
ꢀ1
ꢀ1
ꢀ1
2
957 cm and 2852 cm (curve b); 2947 cm (curve c) are assigned
to C–H stretching vibration of CH –. The characteristic band at 1275
3
ꢀ1
cm (curve a) represented the C–O–C stretching vibration. The C=O
stretching vibration and the C–O–C stretching vibration of pꢀacid are
a
means to prove the successful synthesis of pꢀacid. The
ꢀ1
characteristic band at 3415 cm (curve c) corresponds to the
stretching vibration of –OH group, together with existence of the
C=O stretching vibration. The peaks at 2150 cm (curve b), 2202
cm (curve c) belong to C≡C stretching vibration. From the C≡C
stretching vibration, we can clearly see the difference between pꢀ
ꢀ1
ꢀ1
ꢀ1
acidꢀBr and pꢀacid. The peaks at 1719 cm (curve b) belong to the
ꢀ1
ꢀ1
CꢀBr stretching vibration 3336 cm and 1638 cm (curve c) belong
to the CꢀS stretching vibration. Furthermore, compared with pꢀacidꢀ
Br, pꢀacidꢀBrꢀthiourea has red shifted on C≡C stretching vibration,
we conclude that the pꢀacidꢀBr can compound with thiourea. All of
these preliminary prove that the pꢀacid, pꢀacidꢀBr, pꢀacidꢀBrꢀ
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DOI: 10.1039/C 695
Jou6RrnA0al Na3Cme
1
thiourea were synthesized successfully. Data from H NMR spectra
CꢀNH2 at room temperature. PꢀacidꢀBr has no FL because of the
presence of –Br and C=O were made the FL disappeared, with the
addition of thiourea which reacted with –Br and C=O, so that the FL
appeared. Therefore, this fluorescence sensor could provide a
promising platform for rapid screening and quantification of thiourea
levels. An incubation time of 60 min was selected for sensitive
1
of pꢀacid are shown in Figure SI H NMR (400 MHz, CDCl , ppm):
.26 (2H), 8.00 (4H), 7.65–7.67 (4H), 3.85–3.87 (6H). Elemental
analysis of C O H gave a composition (%) of C 71.05, O 24.33, H
.62, which was in good agreement with the theoretical composition
3
7
26
6
18
4
of C O H : C 73.23, O 22.53, H 4.23.
2
6
6
18
Possible mechanism for FL production of p-acid and p-acid-Br- determination of thiourea in the subsequent experiments.
thiourea
FL is a means of converting optical energy to another optical
energy. It involves the production of reactive intermediates from
stable precursors. These intermediates then react under a variety of
conditions to form excited states emitting light. The mechanism for
the FL production of pꢀacid could be inferred according to the study
of FL. The electrons within the pꢀacid are firstly excited to the
excited state under photoꢀirradiation (hν ), the pꢀacid could be
1
•
+
oxidated to pꢀacid . The electron lost from one pꢀacid molecule can
further react with another pꢀacid molecule forming the strong
oxidant pꢀacid . pꢀacid could react with pꢀacid through electron
transfer generating the excited state of polymer (pꢀacid*) which
•
ꢀ
•ꢀ
•+
could emit light (hν ). All possible FL mechanisms of pꢀacid are
2
described as following equations (1)ꢀ(4):
•
+
ꢀ
hν + pꢀacid → pꢀacid + e
(1)
(2)
(3)
(4)
1
ꢀ
•ꢀ
pꢀacid + e → pꢀacid
Scheme 2 The FL mechanism of thiourea
Optimization of experimental conditions
pꢀacid• + pꢀacid → pꢀacid + pꢀacid
+
•ꢀ
*
*
The time and temperature of the pꢀacidꢀBrꢀthiourea reaction
also greatly affected the analytical performance of the proposed
detection. The effect of the incubation time of thiourea with pꢀacidꢀ
Br (Figure 2A) on the fluorescence intensity of the system is
investigated, from which it can be seen that the optimal incubation
time is 50 minutes. The fluorescence intensity increases with the
increasing of the incubation time, and inclines to a constant value
after 50 minutes, ascribing to the saturated binding between the
thiourea and pꢀacidꢀBr.
pꢀacid → pꢀacid + hν
2
The mechanism between pꢀacidꢀBr and thiourea (Tu), just as in
the mechanism of pꢀacid, in the first step, pꢀacidꢀBr could react with
Tu to form a new compound of pꢀacidꢀBrꢀTu. Then, the electrons
within the pꢀacidꢀBrꢀTu are firstly excited to the excited state under
photoꢀirradiation (hν ), the pꢀacidꢀBrꢀTu could be oxidated to pꢀacidꢀ
1
•
+
BrꢀTu . The electron lost from one pꢀacidꢀBrꢀTu molecule can
further react with another pꢀacidꢀBrꢀTu molecule forming the strong
•
ꢀ
•ꢀ
oxidant pꢀacidꢀBrꢀTu . pꢀacidꢀBrꢀTu could react with pꢀacidꢀBrꢀ
•
+
Furthermore, with an increasing incubation temperature from
Tu through electron transfer generating the excited state of
1
0 °C to 50 °C (Figure 2B), the FL detection after incubation for 50
polymer (pꢀacidꢀBrꢀTu*) which could emit light (hν ). In summary,
2
min with 10 nM thiourea showed a maximum fluorescence response
at 35 °C. To simplify the analytical process, all the experiments were
carried out at room temperature. At this temperature, no further
significant changes were observed in the fluorescence generated.
the possible FL mechanisms between pꢀacidꢀBrꢀTu are described in
equations (5)ꢀ(9):
pꢀacidꢀBr + Tu → pꢀacidꢀBrꢀTu
hν + pꢀacidꢀBrꢀTu → pꢀacidꢀBrꢀTu + e
(5)
(6)
(7)
The influence of the pH value of the detection solution is an
important parameter because the acidity of the solution greatly
affects the activity of the pꢀacidꢀBr. The effect of the solution pH on
the fluorescence responses of the analysis was investigated with 10
nM thiourea. Figure 2C shows the effect of pH value of the detection
solution on the fluorescence change. The fluorescence change was
increased with the increment of pH value from pH 5.5 to 7.4 and
then when the pH value was above 7.4 the fluorescence change
decreased. The optimal fluorescence response was therefore
achieved at pH 7.4. The reason is that highly acidic or alkaline
surroundings would damage the pꢀacidꢀBr, especially in an acidic
environment.
•
+
ꢀ
1
ꢀ
•ꢀ
pꢀacidꢀBrꢀTu + e → pꢀacidꢀBrꢀTu
pꢀacidꢀBrꢀTu• + pꢀacidꢀBrꢀTu →
+
•ꢀ
*
pꢀacidꢀBrꢀTu + pꢀacidꢀBrꢀTu
(8)
(9)
*
pꢀacidꢀBrꢀTu → pꢀacidꢀBrꢀTu + hν
2
The kinetic behavior of pꢀacidꢀBr reacting with thiourea was
studied by monitoring the fluorescence recovery as a function of
time. As shown in Figure 2A and Scheme 2, the fluorescence Analytical Performance
intensity of pꢀacidꢀBr gradually increased with the elongation of
time and reached equilibrium after 60 min, revealing a cyclization
reaction between pꢀacidꢀBr with –Br, C=O and thiourea with C=S,
Figure 3A showed the FL intensity of the solution in the
absence (a) and presence (b~k) of different concentrations of
thiourea. It can be seen that the FL intensity in the presence of
4
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thiourea (b) was higher than that in the absence of thiourea (a), and
the FL intensity increased gradually with increasing concentrations
of thiourea (b~k). The reason was that the specific binding of pꢀacidꢀ
Br and thiourea could produce the FL singal in the solution, and thus
increased the FL intensity, which suggested that the thiourea
concentration could be determined by the FL measurement of this
reaction. The standard calibration curve for the thiourea detection is
shown in Figure 3 B and C. The FL intensity increased linearly with
the thiourea concentration from 0.5 nM to 1000 nM with a detection
limit of 0.26 nM. The linear fit equation is FL = 194.68+ 212.86
lgcthiourea (unit of c is nM), with a correlation coefficient of 0.9889.
According to the linear equation, we could detect trace thiourea
concentration quantitatively. Higher plasma thiourea levels could be
detected by an appropriate dilution with pH 7.4 PBS. To further
clarify the performances of the developed analysis method toward
the pꢀacidꢀBr and thiourea reaction, the analytical performance of the
proposed fluorescence analysis has been compared with those of
other thiourea detection. Characteristics such as the linear range and
detection limit are summarized for all of them in Table 1. As can be
observed, the detection limit of the developed fluorescence method
exhibited a higher sensitivity and lower detection limit than those of
Figure 3 (A) FL profiles of the sensors in the presence of different
ꢀ
1
concentrations of thiourea in PBS containing 1 mol⋅L DMF, pH
.4. (B) Relationship between FL and thiourea concentration each
7
point is the average of three measurements. (C) Calibration curve for
thiourea determination, thiourea determination (nM): (a) 0, (b) 0.5,
(c) 1, (d) 5, (e) 10, (f) 50, (g) 85, (h) 140, (i) 200, (j) 500, (k) 1000.
other method. The reason might be the fact that the pꢀacidꢀBrꢀ
thiourea has a high quantum yield. Importantly, the high quantum
yield could make it especially suitable for ultrasensitive bioanalysis
without the need for additional reagents or signal amplification steps.
Specificity of co-existing molecules and ions on the detection of
thiourea
To investigate the selectivity and specificity of the developed
method for detecting thiourea, interference study was employed.
Under the same assay conditions, the FL value of thiourea, some
common molecules and inorganic ions were measured using the asꢀ
prepared pꢀacidꢀBr probe, respectively. In this section, the
measurands were chosen as 100 nM thiourea, 100 mM xylose, 100
mM glucose, 100 mM citric acid, 200 mM urea. As shown in Figure
4
A, the FL value of thiourea was much higher than that of the other
measurands, even when their concentration was at least ten times
higher than that of thiourea. Thus, this developed method exhibits
outstanding selectivity for detecting thiourea. In order to study the
interfering effects or selectivity of the procedure, many of the
coexisting species in real waters should be investigated. To evaluate
the antiꢀinterference ability of the asꢀdeveloped method for detecting
thiourea, an assay was carried out by measuring the FL of the pꢀacidꢀ
Br probe for detecting 100 nM thiourea with a series of coꢀexisting
2
ꢀ
+
+
ꢀ
ions, including 10 mM S O , 10 mM K , 10 mM NH , 10 mM Cl ,
2
3
4
ꢀ
2ꢀ
ꢀ
2+
+
NO , SO , CH COO , Ca and Na , respectively. It revealed that
3
4
3
the FL value of the pꢀacidꢀBr for detecting thiourea was negligibly
reduced by the coꢀexisting molecules and ions with much higher
concentration than that of thiourea, although the concentration of
urea was 2000 times higher that of thiourea (Figure 4B). This result
indicated that the developed method shows an excellent performance
for antiꢀinterference in the detection of thiourea with the presence of
so much coꢀexisting molecules and ions. when the various coexisting
species were added to the thourea solution, the FL intensity was
almost not changed because they do not have the group of C=S, Cꢀ
Figure 2 Effect of reaction time (A), reaction temperature (B),
and pH (C) on the FL intensity.
Table 1 The results of comparing with other reported methods
for the linear range and LOD of thiourea.
Method
Linear range /nM
LOD /nM
13.1
Ref.
38
2ꢀ
+
+
ꢀ
ꢀ
2ꢀ
ꢀ
2+
+
NH in S O , K , NH , Cl , NO , SO , CH COO , Ca and Na .
2
2
3
4
3
4
3
2
4
Mass spectrometry
1.3×10 ~6.6×10
So that they were just revealing a cyclization reaction between pꢀ
acidꢀBr with –Br, C=O and thiourea with C=S, CꢀNH at room
2
5
7
5
2ꢀ
+
+
ꢀ
ꢀ
2ꢀ
ꢀ
2+
FTIR
7.9×10 ~1.1×10
1.3×10
39
40
temperature, S O , K , NH , Cl , NO , SO , CH COO , Ca and
2
3
4
3
4
3
+
Na were not revealing a cyclization reaction with pꢀacidꢀBr. So that
pꢀacidꢀBr can specifically react with thiourea. Hence this method
has a high specificity to detect thiourea and can be further applied
for real samples.
3
Chemiluminescence
10.0~1.0×10
10.0
Catalytic kinetic
spectrophotometry
2
5
2
3
.9×10 ~1.3×10
2.6×10
41
42
2
Spectrophotometry
Fluorescence
5.0~2.3×10
2.14
0.26
3
This
work
0.5~1.0×10
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1-3 | 5

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ARTICLE
DOI: 10.1039/C 695
Jou6RrnA0al Na3Cme
Analytical application for determination of thiourea in real
samples
Preparation of real samples. According to the applications of
thiourea in industry and agriculture mentioned before, water samples
(DI water sample, two industrial waste water samples, one river
water sample) and juice samples (one orange juice, one lemon juice
and one apple juice) were collected as the real samples. Both of the
two industrial waste water samples were filtered with 0.22 µm
micropore membranes and diluted to 10 folds. Juice samples were
obtained by squeezing fruits. The three fruit samples were purchased
from a local market in Jinan. The collected samples were rinsed with
tap water, distilled water, and ultrapure water, three times
respectively, and then dried and stored in clean beakers at room
temperature for several hours. After drying, fruits were squeezed to
obtain juices. The juice samples were centrifuged for 15 min with
5000 rpm. The supernatant liquid was filtered by 0.22 µm micropore
membranes. To remove the excess of ascorbic acid, the juice
samples were stirred for 10 min. Before analysis, the juice samples
Figure 4 (A) is the specificity test and (B) is the coꢀexisting test.
Table 2 Determination of thiourea in real samples.
Concentration
Concentration
of added
Concentration
found /nm
found after
added
Recovery
/%
Sample
thiourea /nm
thiourea /nm
42,43
DI
none
ꢀꢀ
ꢀꢀ
ꢀꢀ
were diluted to 100 folds.
immediately after sampling.
All of samples were analyzed
water
Procedure for the detection of thiourea. With high specificity
and selectivity in connection with thiourea leaching pꢀacidꢀBr, the
probe exhibited a good performance in the detection of thiourea, as
supported by the neglectable changes of the FL in the presence of
interference. The method in this work was applied for real samples
such as water samples (doubleꢀdistilled water sample, two industrial
waste water samples, one river water sample) and juice samples (one
orange juice, one lemon juice and one apple juice), as shown in
Figure 5A. 1 mL PBS (pH 7.4) and added with 500 µL of pꢀacidꢀBr
1
1
1
1
1
1
31.4
31.4
31.4
06.6
06.6
06.6
ꢀꢀ
ꢀꢀ
ꢀꢀ
Waste
100
300
ꢀꢀ
228.3
305.2
ꢀꢀ
98.7
101.7
ꢀꢀ
water 1
Waste
ꢀ1
solution (dissolved in PBS containing 1 mol⋅L DMF, pH7.4). Then
100
300
ꢀꢀ
210.4
289.3
ꢀꢀ
101.8
96.4
ꢀꢀ
water 2
different concentrations thiourea solution was added to the system.
About 60 min later, the FL absorbance peak of the solution was
stable, we signed the FL1 intensity. The absorbance of the blank
sample was determined using the same procedure without the
none
none
none
none
none
none
none
none
none
none
none
none
addition of thiourea and signed as FL . The different of FL
0
intensities (FL ꢀFL ) of the solution was recorded to quantify the
1
0
River
water
100
300
ꢀꢀ
98.6
302.4
ꢀꢀ
98.6
100.8
ꢀꢀ
concentration of thiourea. The thiourea was determined in two
industrial waste water samples. No thiourea could be detected in the
natural water samples and fruit juice samples. The recoveries of
thiourea at the 100 nM level and 300 nM level, respectively. The
results were shown in Table 2. From Table 2 we can calculate the
standard deviations (RDs), RDs of waste water 1 were ꢀ1.36 and
1.10, RDs of waste water 2 were 1.81 and 3.44, RDs of river water
were ꢀ1.42 and 0.79, RDs of orange juice were 2.06 and 0.60, RDs
of apple juice were ꢀ0.91 and 0.7, RDs of lemon juice were 0.9 and ꢀ
Orange
juice
100
300
ꢀꢀ
102.1
301.8
ꢀꢀ
102.1
100.6
ꢀꢀ
1
.67. These results indicated that the standard deviations are all less
than 4.00%, which suggested that the specificity of the asꢀprepared
FL sensor was acceptable. Therefore, this method could be
reasonably applied in the environmental detection of thiourea.
Apple
juice
100
300
ꢀꢀ
99.1
302.2
ꢀꢀ
99.1
100.7
ꢀꢀ
In an attempt to test the applicability of our FL sensor for
detection in biological sample matrixe, we employed diluted human
serum samples (1:10). We added with 500 µL of pꢀacidꢀBr solution
ꢀ
1
(dissolved in PBS containing 1 mol⋅L DMF, pH7.4) into the diluted
human serum. Then different concentrations of thiourea solutions
were added to the system. About 60 min later, the FL absorbance
peak of the solution was stable, founding there was no thiourea in
human serum samples. The different of FL intensities of the solution
was recorded with adding different concentrations of thiourea (as
shown in Figure 5B). Therefore, this method could be reasonably
applied in the biological sample matrixes detection of thiourea.
Lemon
juice
100
300
100.9
298.1
100.9
99.4
6
| J. Name., 2012, 00, 1-3
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ART53ICLE
DOI: 10.1039/C6RA069 C
4
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Figure 5 (A) Real samples detection, (B) Human serum
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0 nM, (c) 100 nM, (d) 150 nM.
1
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Analyst 2011, 136, 5256.
In summary,
a novel organic fluorescence system was
1
1
4 Y.A. Akeneev, E.A. Zakharova, G.B. Slepchenko and N.P. Pikula, J.
Anal. Chem. 2005, 60, 514ꢀ517.
developed to detect thiourea based on pꢀacidꢀBr. Compared
with other methods, this method has advantages such as a
simple and fast detection process, high sensitivity and
specificity, and powerful tolerance to complex coexisting
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determination in various water samples and fruit juice samples
available in the local market, and thus provide a new
promising platform for clinical analysis. Furthermore, it will
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1
is to open new avenues in the application of phenyleneꢀ 19 K. Kargosha, M. Khanmohammadi and M. Ghadiri, Analyst, 2001, 126,
ethynylene derivative for sensitive thiourea assay. Hence, the
proposed fluorescence sensor could become a promising
method for local market.
1432.
2
2
2
2
0 J. Rethmeier, G. Neumann, C. Stumpf, A. Rabenstein and C.Vogt, J.
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This work was financially supported by National Natural
Science Foundation of China (51273084, 51473067), Shandong
Provincial Excellent Youth Fund (ZR2015JL019).
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DOI: 10.1039/C6RA0695
Journal Na3Cme
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DOI: 10.1039/C6RA06953C
Graphical Abstract
A novel organic fluorescence system was developed to detect thiourea based on
p-acid-Br, which in the range of 0.5~1000 nM and with a detection limit of 0.26 nM.
And this method provides a new promising platform for clinical analysis.