U.S. patent application number 16/612416 was filed with the patent office on 2021-02-18 for method for preparing ratiometric fluorescent probe for melamine based on silver nanocluster complex.
This patent application is currently assigned to Qingdao University. The applicant listed for this patent is Qingdao University. Invention is credited to Xiangning BU, Yongxin FU, Rijun GUI, Hui JIN.
Application Number | 20210047559 16/612416 |
Document ID | / |
Family ID | 1000005370931 |
Filed Date | 2021-02-18 |
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United States Patent
Application |
20210047559 |
Kind Code |
A1 |
GUI; Rijun ; et al. |
February 18, 2021 |
METHOD FOR PREPARING RATIOMETRIC FLUORESCENT PROBE FOR MELAMINE
BASED ON SILVER NANOCLUSTER COMPLEX
Abstract
A method for preparing a ratiometric fluorescent probe for
melamine based on a DNA-stable silver nanocluster-rhodamine 6G
complex, wherein the electrostatic self-assembly technology is
adopted to construct a silver nanocluster-rhodamine 6G complex. The
melamine forms a strong hydrogen bond with thymine in the DNA of
the surface of the silver nanocluster, causing rhodamine 6G to
dissociate from the surface of the silver nanocluster, destroying
the fluorescence resonance energy transfer, so as to restore the
fluorescence of the silver nanocluster. This process has little
effect on Rhodamine 6G fluorescence which can be used as a
reference signal, while silver nanocluster fluorescence can be used
as a response signal. By fitting the linear relationship between
the ratio of fluorescence emission peak intensities of the silver
nanocluster to rhodamine 6G and the molar concentration of the
melamine, the ratiometric fluorescent probe for melamine can be
constructed.
Inventors: |
GUI; Rijun; (Qingdao,
CN) ; FU; Yongxin; (Qingdao, CN) ; JIN;
Hui; (Qingdao, CN) ; BU; Xiangning; (Qingdao,
CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Qingdao University |
Qingdao |
|
CN |
|
|
Assignee: |
Qingdao University
Qingdao
CN
|
Family ID: |
1000005370931 |
Appl. No.: |
16/612416 |
Filed: |
March 5, 2019 |
PCT Filed: |
March 5, 2019 |
PCT NO: |
PCT/CN2019/076942 |
371 Date: |
November 11, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C09K 2211/1029 20130101;
C09K 2211/1007 20130101; C09K 11/06 20130101; G01N 2021/6432
20130101; G01N 21/643 20130101; C09K 11/58 20130101; C07D 311/78
20130101 |
International
Class: |
C09K 11/06 20060101
C09K011/06; C07D 311/78 20060101 C07D311/78; C09K 11/58 20060101
C09K011/58; G01N 21/64 20060101 G01N021/64 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 13, 2018 |
CN |
201810604913.2 |
Claims
1. A method for preparing a ratiometric fluorescent probe for
melamine based on a silver nanocluster complex, comprising the
following steps: (1) preparation of DNA-stable silver nanoclusters:
at 0.degree. C., adding a predetermined amount of silver nitrate
solution and DNA solution to 1 mL of double-distilled water to
obtain a first solution, stirring the first solution magnetically
for 20 minutes to form a homogeneous mixture, then adding a sodium
borohydride solution to the homogeneous mixture to obtain a second
solution, and performing a reaction on the second solution under a
vigorous stirring in a dark place for 20 minutes to obtain a first
product solution; wherein the first product solution is filtered by
a 0.4 .mu.m filter to obtain a filtrate, and the filtrate is
dialyzed through a dialysis bag with a molecular weight cut-off of
5000 Daltons to remove unreacted experimental materials; a solution
in the dialysis bag is subjected to a rotary evaporation to remove
90% of a solvent, and then freeze-dried to obtain a dry sample of
the DNA-stable silver nanoclusters; the dry sample of the
DNA-stable silver nanoclusters is stored at 4.degree. C. in a dark
condition; (2) preparation of a DNA-stable silver
nanocluster-rhodamine 6G complex: dissolving the DNA-stable silver
nanoclusters prepared in step (1) in 200 .mu.L of double-distilled
water to obtain a third solution, adding 100 .mu.L of citrate
buffer to the third solution to mix well to obtain a fourth
solution, and then adding 100 .mu.L of rhodamine 6G solutions with
different molar concentrations to the fourth solution to obtain a
plurality of fifth solutions; wherein each fifth solution of the
plurality of fifth solutions reacts in a dark place for 30 minutes
to obtain a second product solution, and the second product
solution is subjected to centrifugal separation, ethanol washing
and vacuum drying to obtain the DNA-stable silver
nanocluster-rhodamine 6G complex; (3) dispersing the DNA-stable
silver nanocluster-rhodamine 6G complex prepared in step (2) in 100
.mu.L of citrate buffer to obtain a sixth solution, incubating the
sixth solution in a dark place for 30 minutes to obtain a first
homogeneous solution, measuring a fluorescence emission spectrum of
the first homogeneous solution, optimizing an intensity of a double
emission fluorescence peak of the first homogeneous solution to
determine a ratio of the DNA-stable silver nanoclusters and the
rhodamine 6G to obtain a homogeneous solution having an optimized
ratio; (4) at room temperature and under a slow magnetic stirring,
adding 15 .mu.L of melamine solutions with different concentrations
to the homogeneous solution having the optimized ratio to obtain a
plurality of seventh solutions, continuing to stir the plurality of
seventh solutions for 5 minutes to fully react to form a plurality
of second homogeneous solutions, measuring fluorescence emission
spectra of the plurality of second homogeneous solutions, fitting a
linear relationship between a ratio of fluorescence emission peak
intensities of the DNA-stable silver nanoclusters to the rhodamine
6G and a molar concentration of the melamine, constructing the
ratiometric fluorescent probe for melamine; wherein in step (1), a
size of each of the DNA-stable silver nanoclusters is 6-12 nm, a
molar concentration of the silver nitrate solution is 5-10 mmol/L,
a molar concentration of the DNA solution is 200-600 mmol/L, and a
molar concentration of the sodium borohydride solution is 5-10
mmol/L; in step (2), a pH of the citrate buffer is 5.5-6.5, and a
range of the different molar concentrations of the rhodamine 6G is
0.1-2 nmol/L; in step (3), a mass concentration of the sixth
solution is 1-5 mg/mL; in step (4), a range of the different
concentrations of the melamine solutions is 0.5-15 .mu.mol/L, and a
detection limit of the melamine is 0.05-0.2 .mu.mol/L.
Description
CROSS REFERENCE TO THE RELATED APPLICATIONS
[0001] This application is the national phase entry of
International Application No. PCT/CN2019/076942, filed on Mar. 5,
2019, which is based upon and claims priority to Chinese Patent
Application No. 201810604913.2, filed on Jun. 13, 2018, the entire
contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present disclosure belongs to the technical field of
preparations of noble metal nanomaterials and ratiometric
fluorescent probes, and more specifically relates to a method for
preparing a ratiometric fluorescent probe for melamine based on a
DNA-stable silver nanocluster-rhodamine 6G complex and the probe
prepared by the method can be used for a high sensitivity detection
of melamine.
BACKGROUND
[0003] Melamine, a heterocyclic organic compound containing
nitrogen with a nitrogen content of up to 66%, is commonly used in
plastics, pesticides, fertilizers and other industries. Adding 1%
of melamine can increase a protein content by more than 4% in food,
and some companies use melamine to replace protein for high
profits. The U.S. Food and Drug Administration and Ministry of
Health of the People's Republic of China stipulate a safety limit
of 1 mg/kg for infant food and 2.5 mg/kg for adult food. Detecting
the content of melamine in food is important for the health of
consumer. Present methods of detecting melamine include high
performance liquid chromatography, gas chromatography and mass
spectrometry, spectrophotometry, surface enhanced Raman scattering
and so on. These traditional detection methods generally have
problems such as time consumption during sample preparation,
complex operation, expensive instrumentation, and high detection
cost. Therefore, developing a low-cost, simple, fast and efficient
method for detecting melamine has become a key technical object to
be attained urgently.
[0004] Fluorimetry is an analytical method for substance
identification and content determination based on a change of
fluorescence intensity or spectral line position caused by reaction
of substances or their surface modified functional groups with
substances for performing a detection. The method has the
advantages of simple operation and high sensitivity. Literatures
are searched as follows. Wu et al. used fluorescence resonance
energy transfer between up-conversion nanoparticles and gold
nanoparticles to detect melamine (An upconversion fluorescence
resonance energy transfer nanosensor for one step detection of
melamine in raw milk, Qiongqiong Wu, Qian Long, Haitao Li, Youyu
Zhang, Shouzhuo Yao, Talanta, 2015, 136, 47-53). Kalaiyarasa et al.
designed a melamine fluorescence sensor based on gold nanoclusters
(Melamine dependent fluorescence of glutathione protected gold
nanoclusters and ratiometric quantification of melamine in
commercial cow milk and infant formula, Gopi Kalaiyarasan, Anusuya
K, James Joseph, Appl. Surf Sci., 2017, 420, 963-969). Cuifeng
Jiang et al. reported a method for detecting melamine based on
two-photon excitation fluorescence (Patent Publication No.
CN105158225A).
[0005] Ratiometric fluorescence method is a method of quantitative
detection based on a ratio of fluorescence intensities at two
different wavelengths by a reaction of dual-emission fluorescent
probe with a substance to be detected. Ratiometric fluorescence
method has a self-calibration function, which can eliminate a
fluorescence interference generated by the system itself and
environmental factors, and effectively improve the accuracy and
reliability of the detection result of a target object. Silver
nanoclusters have unique physicochemical properties, such as strong
fluorescence emission, good photostability, high biocompatibility,
and sub-nano size, and can be used in nanomedicine, bioimaging,
drug delivery, biochemical sensing and many other fields. Rhodamine
6G is a widely used organic dye with the advantages of high
photostability, pH insensitivity and high fluorescence quantum
yield. Although the literature on melamine fluorescence detection
has been reported, the present inventors have first constructed a
dual-emission fluorescent probe based on a DNA-stable silver
nanocluster-rhodamine 6G complex, and applied the dual-emission
fluorescent probe to a ratiometric fluorescence detection of
melamine in actual samples. So far, the ratiometric fluorescent
probe for melamine based on DNA-stable silver nanocluster-rhodamine
6G complex has not yet been reported in domestic and foreign
literature or patent.
SUMMARY
[0006] The objective of the present disclosure is to overcome the
deficiencies of the prior art described above, and to design a
ratiometric fluorescent probe for melamine based on a DNA-stable
silver nanocluster-rhodamine 6G complex, where the ratiometric
fluorescent probe is simple, easy to prepare, low in cost and high
in sensitivity.
[0007] In order to achieve the aforementioned objective, the
present disclosure provides a method for preparing a ratiometric
fluorescent probe for melamine based on a silver nanocluster
complex, and a preparation process thereof includes the following
steps.
[0008] (1) Preparation of DNA-stable silver nanoclusters: at
0.degree. C., adding a certain amount of silver nitrate solution
and DNA solution to 1 mL of double-distilled water, stirring
magnetically for 20 minutes to form a homogeneous mixture, then
adding a freshly prepared sodium borohydride solution, and reacting
under a vigorous stirring in a dark place for 20 minutes. The
resulting product solution is filtered by a 0.4 .mu.m filter, and a
filtrate is dialyzed through a dialysis bag with a molecular weight
cut-off of 5000 Daltons to remove unreacted experimental materials.
The solution in the dialysis bag is subjected to a rotary
evaporation to remove 90% of the solvent, and then freeze-dried to
obtain a dry sample of the silver nanoclusters. The dry sample of
the silver nanoclusters is stored at 4.degree. C. in a dark
condition.
[0009] (2) Preparation of silver nanocluster-rhodamine 6G complex:
dissolving the silver nanoclusters prepared in step (1) in 200
.mu.L of double-distilled water, adding 100 .mu.L of citrate buffer
to mix well, and then adding 100 .mu.L of rhodamine 6G solution
with different concentrations. The mixed solution reacts in a dark
place for 30 minutes, and the product solution is subjected to
centrifugal separation, ethanol washing and vacuum drying to obtain
the silver nanocluster-rhodamine 6G complex.
[0010] (3) Dispersing the complex prepared in step (2) in 100 .mu.L
of citrate buffer, incubating in a dark place for 30 minutes,
measuring fluorescence emission spectra of homogeneous solution of
complex corresponding to different molar concentrations of the
rhodamine 6G, optimizing the intensity of the double emission
fluorescence peak to determine the ratio of silver nanoclusters and
rhodamine 6G.
[0011] (4) At room temperature and under a slow magnetic stirring,
adding 15 .mu.L of melamine solutions with different concentrations
to a homogeneous solution of optimized ratio complex prepared in
step (3), continuing to stir for 5 minutes to fully react to form a
homogeneous solution of the complex and the melamine, measuring
fluorescence emission spectra of the homogeneous solution of the
complex and melamine corresponding to different molar
concentrations of melamine, fitting linear relationship between the
ratio of the fluorescence emission peak intensities of the silver
nanoclusters to the rhodamine 6G and the molar concentration of the
melamine, constructing the ratiometric fluorescent probe for
melamine.
[0012] In step (1) of the present disclosure, the size of the
silver nanocluster is 6-12 nm, and molar concentrations of the
silver nitrate, the DNA and the sodium borohydride are 5-10 mmol/L,
200-600 .mu.mol/L and 5-10 mmol/L, respectively. In step (2), pH of
the citrate buffer is 5.5-6.5, and the molar concentration of
rhodamine 6G is 0.1-2 nmol/L. In step (3), the mass concentration
of the complex solution is 1-5 mg/mL. In step (4), the
concentration range of the melamine is 0.5-15 mol/L, and the
detection limit is 0.05-0.2 mol/L.
[0013] Compared with the prior art, the present disclosure employs
an electrostatic self-assembly technology to construct a DNA-stable
silver nanocluster-rhodamine 6G complex. The absorption spectrum of
rhodamine 6G partially overlaps with the fluorescence emission
spectrum of silver nanocluster, the fluorescence resonance energy
transfer occurs between the two and causes the fluorescence
quenching of silver nanocluster, while the fluorescence of
rhodamine 6G is enhanced. The added melamine can form a strong
hydrogen bond with thymine on the surface of the DNA-stable silver
nanocluster, causing rhodamine 6G to dissociate from the surface of
the silver nanocluster, increasing the distance between the donor
and the receptor, and weakening the fluorescence resonance energy
transfer, so as to restore the fluorescence of the silver
nanocluster. This process has little effect on Rhodamine 6G
fluorescence which can be used as a reference signal, while silver
nanocluster fluorescence can be used as a response signal. By
fitting the linear relationship between the ratio of the
fluorescence emission peak intensities of the silver nanocluster to
the rhodamine 6G and the molar concentration of the melamine, the
ratiometric fluorescent probe for melamine can be constructed. The
probe has simple preparation process, low cost and high product
sensitivity, and can be developed into a novel ratiometric
fluorescent probe for melamine, where the novel ratiometric
fluorescent probe is suitable for efficiently detecting melamine in
actual samples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 is a schematic diagram of a method for preparing
ratiometric fluorescent probe for melamine based on a DNA-stable
silver nanocluster-rhodamine 6G complex in the present
disclosure;
[0015] FIG. 2 is a graph showing response to fluorescence emission
peak intensity of DNA-stable silver nanocluster-rhodamine 6G
complex as molar concentration of rhodamine 6G increases;
[0016] FIG. 3 is a graph showing response to fluorescence emission
peak intensity of DNA-stable silver nanocluster-rhodamine 6G
complex as molar concentration of melamine increases;
[0017] FIG. 4 is a graph showing a fitted linear relationship
between the ratio of fluorescence emission peak intensities of
DNA-stable silver nanocluster to rhodamine 6G and molar
concentration of melamine.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0018] The present disclosure will be further described below in
conjunction with the drawings and specific embodiments.
Embodiment 1
[0019] This embodiment relates to a method for preparing a
ratiometric fluorescent probe for melamine based on a DNA-stable
silver nanocluster-rhodamine 6G complex. The preparation process of
the ratiometric fluorescent probe for melamine and the principle of
the ratiometric fluorescence detection of melamine are shown in
FIG. 1, and the specific process steps are as follows.
[0020] Preparation of DNA-stable silver nanoclusters: at 0.degree.
C., a certain amount of silver nitrate solution and DNA solution
are added to 1 mL of double-distilled water, a magnetic stirring is
performed for 20 minutes to form a homogeneous mixture, then a
freshly prepared sodium borohydride solution is added, and a
reaction is carried under a vigorous stirring in a dark place for
20 minutes, wherein molar concentrations of the silver nitrate, the
DNA and the sodium borohydride are 5 mmol/L, 200 .mu.mol/L and 5
mmol/L, respectively. The resulting product solution is filtered by
a 0.4 .mu.m filter, and a filtrate is dialyzed through a dialysis
bag with a molecular weight cut-off of 5000 Daltons to remove
unreacted experimental materials, and the solution in the dialysis
bag is subjected to a rotary evaporation to remove 90% of the
solvent, and then freeze-dried to obtain a dry sample of the silver
nanoclusters, and the dry sample of the silver nanoclusters is
stored at 4.degree. C. in a dark condition.
[0021] Preparation of silver nanocluster-rhodamine 6G complex: the
prepared silver nanoclusters are dissolved in 200 .mu.L of
double-distilled water, 100 .mu.L of citrate buffer (pH 5.5) is
added to mix well, and then 100 .mu.L of rhodamine 6G solution
(0.1-0.5 nmol/L) is added. The mixed solution reacts in a dark
place for 30 minutes, and the product solution is subjected to
centrifugal separation, ethanol washing and vacuum drying to obtain
the silver nanocluster-rhodamine 6G complex.
[0022] The prepared complex is dispersed in 100 .mu.L of citrate
buffer, and the mass concentration of the complex is 1-2 mg/mL.
After homogeneous solution of the complex is incubated in a dark
place for 30 minutes, fluorescence emission spectra of the
homogeneous solution of the complex corresponding to different
molar concentrations of rhodamine 6G are measured respectively, and
the intensity of double emission fluorescence peak is optimized to
determine the ratio of silver nanocluster and rhodamine 6G (see
FIG. 2).
[0023] At room temperature and under a slow magnetic stirring, 15
.mu.L of melamine solutions with different concentrations are added
to the prepared homogeneous solution of complex having the
optimized ratio, and the solution is continuesly stirred for 5
minutes to fully react to form a homogeneous solution of the
complex and the melamine. Fluorescence emission spectra of the
homogeneous solution of the complex and melamine corresponding to
different molar concentrations of melamine are measured
respectively (see FIG. 3). The ratio F.sub.DNA-Ag NCs/F.sub.Rh 6G
of fluorescence emission peak intensities of the silver nanocluster
to rhodamine 6G and the molar concentration CMA of the melamine are
fitted to obtain a linear relationship as F.sub.DNA-Ag NCs/F.sub.Rh
6G=0.06014C.sub.MA+0.5612 (R.sup.2=0.9959) (see FIG. 4). Therefore,
the ratiometric fluorescent probe for melamine can be constructed,
wherein the linear concentration of melamine to be detected is 2-10
.mu.mol/L, and the detection limit of melamine is 0.2
.mu.mol/L.
Embodiment 2
[0024] The specific process steps for preparing DNA-stable silver
nanoclusters in this embodiment are the same as those in embodiment
1, wherein the molar concentrations of silver nitrate, DNA and
sodium borohydride are 8 mmol/L, 400 .mu.mol/L and 7 mmol/L,
respectively. The prepared silver nanoclusters are dissolved in 200
.mu.L of double-distilled water, 100 .mu.L of citrate buffer (pH
6.0) is added to mix well, and then 100 .mu.L of rhodamine 6G
solution (0.2-1 nmol/L) is added. The mixed solution reacts in a
dark place for 30 minutes, and the product solution is subjected to
centrifugal separation, ethanol washing and vacuum drying to obtain
a silver nanocluster-rhodamine 6G complex. The prepared complex is
dispersed in 100 .mu.L of citrate buffer, and the mass
concentration of the complex is 1-4 mg/mL. After the homogeneous
solution of the complex is incubated in a dark place for 30
minutes, fluorescence emission spectra of the homogeneous solution
of the complex corresponding to different molar concentrations of
rhodamine 6G are measured respectively, and the intensity of double
emission fluorescence peak is optimized to determine the ratio of
silver nanocluster and rhodamine 6G. At room temperature and under
a slow magnetic stirring, 15 .mu.L of melamine solutions with
different concentrations are added to the prepared homogeneous
solution of complex having the optimized ratio, and the solution is
continuously stirred for 5 minutes to fully react to form a
homogeneous solution of the complex and the melamine. Fluorescence
emission spectra of the homogeneous solution of the complex and
melamine corresponding to different molar concentrations of
melamine are measured respectively. By fitting a linear
relationship between the ratio of fluorescence emission peak
intensities of the silver nanocluster to rhodamine 6G and the molar
concentration of the melamine, the ratiometric fluorescent probe
for melamine can be constructed, wherein the linear concentration
of melamine to be detected is 0.5-10 mol/L, and the detection limit
of melamine is 0.05 .mu.mol/L.
Embodiment 3
[0025] The specific process steps for preparing DNA-stable silver
nanoclusters in this embodiment are the same as those in embodiment
1, wherein the molar concentrations of silver nitrate, DNA and
sodium borohydride are 10 mmol/L, 600 .mu.mol/L and 10 mmol/L,
respectively. The prepared silver nanoclusters are dissolved in 200
.mu.L of double-distilled water, 100 .mu.L of citrate buffer (pH
6.5) is added to mix well, and then 100 .mu.L of rhodamine 6G
solution (0.2-2 nmol/L) is added. The mixed solution reacts in a
dark place for 30 minutes, and the product solution is subjected to
centrifugal separation, ethanol washing and vacuum drying to obtain
a silver nanocluster-rhodamine 6G complex. The prepared complex is
dispersed in 100 .mu.L of citrate buffer, and the mass
concentration of the complex is 2-5 mg/mL. After the homogeneous
solution of the complex is incubated in a dark place for 30
minutes, fluorescence emission spectra of the homogeneous solution
of the complex corresponding to different molar concentrations of
rhodamine 6G are measured respectively, and the intensity of double
emission fluorescence peak is optimized to determine the ratio of
silver nanocluster and rhodamine 6G. At room temperature and under
a slow magnetic stirring, 15 .mu.L of melamine solutions with
different concentrations are added to the prepared homogeneous
solution of complex having the optimized ratio, and the solution is
continues stirred for 5 minutes to fully react to form a
homogeneous solution of the complex and the melamine. Fluorescence
emission spectra of the homogeneous solution of the complex and the
melamine corresponding to different molar concentrations of
melamine are measured respectively. By fitting a linear
relationship between the ratio of fluorescence emission peak
intensities of the silver nanocluster to rhodamine 6G and the molar
concentration of the melamine, the ratiometric fluorescent probe
for melamine can be constructed, wherein the linear concentration
of melamine to be detected is 2-15 .mu.mol/L, and the detection
limit of melamine is 0.1 .mu.mol/L.
Embodiment 4
[0026] This embodiment relates to an application of the ratiometric
fluorescent probe for melamine prepared in embodiment 1 in
detecting melamine in an actual sample such as milk. The milk is
mixed with melamine with different concentrations and acetonitrile,
and after 30 minutes of ultrasonic treatment, the mixed solution is
obtained. The mixed solution is centrifuged for 15 minutes at a
speed of 14000 rpm, and the supernatant is taken and filtered. The
filtrate is further diluted 25 times and collected for further
detection. Specifically, for melamine, the detection range of molar
concentration is 0.5-15 .mu.mol/L, the detection limit is 0.09
.mu.mol/L, detection recovery rate is 99.8-100.5%, and relative
standard deviation is 1.1-2.1%. Compared with the prior art, the
previous literatures (Talanta, 2015, 136, 47-53 and Appl. Surf
Sci., 2017, 420, 963-969) use single method or ratiometric
fluorescence method to detect melamine, and the detection recovery
rate of melamine in milk samples is 94.0-102.0%, and the relative
standard deviation is 1.2-3.2%. The ratiometric fluorescent probe
of the present disclosure has high detection recovery rate, low
relative standard deviation, simple preparation process, low cost
and high product sensitivity, and can be developed into a novel
ratiometric fluorescent probe for melamine, where the novel
ratiometric fluorescent probe is suitable for efficiently detecting
melamine in different actual samples.
* * * * *