CN113979996A - Phthalimide fluorescent probe for detecting copper ions, preparation method and application thereof, and copper ion detection method - Google Patents
Phthalimide fluorescent probe for detecting copper ions, preparation method and application thereof, and copper ion detection method Download PDFInfo
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Abstract
The invention belongs to the technical field of organic synthesis and analytical chemistry, and particularly relates to a phthalimide fluorescent probe for detecting copper ions, a preparation method and application thereof, and a copper ion detection method. The fluorescent probe LXJ-3 provided by the invention is a phthalimide compound taking pyridine-2-formate as a recognition receptor, and is a phthalimide derivative. The preparation method comprises the steps of taking N-butyl-3-hydroxyphthalimide and pyridine-2-formyl chloride as raw materials, and carrying out acylation reaction under the catalysis of alkali and 4-dimethylaminopyridine to obtain the fluorescent probe. The method can prepare the fluorescent probe LXJ-3 through two steps, and the synthesis process is simple.
Description
Technical Field
The invention belongs to the technical field of organic synthesis and analytical chemistry, and particularly relates to a phthalimide fluorescent probe for detecting copper ions, a preparation method and application thereof, and a copper ion detection method.
Background
Copper is the third most abundant transition metal element in the human body and widely distributed in the blood and tissues of the human body. Cu2+Plays an important role in physiological processes such as energy generation, signal transduction, oxygen transport and activation of cells. Cu in human body2+Abnormal levels can cause a series of diseases such as Mengkin's disease, Wilson's disease, Alzheimer's disease, Parkinson's disease, coronary heart disease, amyotrophic lateral sclerosis, myelopathy, and the like. Furthermore, Cu is widely used in agricultural and industrial production2+Has become an important environmental pollutant. The U.S. Environmental Protection Agency (EPA) has treated Cu in drinking water2+Is set to 20 μ M. Therefore, the development of a method for detecting Cu with high sensitivity and selectivity2+The method of (a) is very important for human health and environmental safety.
Currently detecting Cu2+The method of (3) includes atomic absorption spectrometry, atomic emission spectrometry, inductively coupled plasma mass spectrometry, electrochemical method, and the like. However, these methods have the disadvantages of time consumption, tedious sample preparation, expensive instruments and unsuitability for real-time analysis. Compared with the method, the fluorescent probe has the advantages of low cost, simple and convenient operation, real-time analysis, nondestructive biological imaging and the like, and is paid more and more attention in the detection of heavy metal ions in recent years. To date, the literature reports a number of Cu2+Fluorescent probes, but many of them still have some limitations, such as poor selectivity, organic solvent requirement, complicated synthesis steps, fluorescence quenching, etc. Therefore, a Cu having good water solubility, simple synthesis and excellent performance has been developed2+Fluorescent probes have important practical significance.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a phthalimide fluorescent probe for detecting copper ions, a preparation method and application thereof, and a copper ion detection method. The fluorescent probe LXJ-3 provided by the invention has the advantages of simple synthesis steps, large Stokes displacement, good water solubility and the like, is an excellent fluorescence-enhanced probe, and has good application prospect.
The technical scheme provided by the invention is as follows:
a phthalimide-based fluorescent probe for detecting copper ions, the fluorescent probe having the following structure:
the fluorescent probe LXJ-3 provided by the technical scheme is a phthalimide compound taking pyridine-2-formate as a recognition receptor, and is a phthalimide derivative.
The invention also provides a preparation method of the phthalimide fluorescent probe for detecting the copper ions, which comprises the following steps:
1) dissolving N-butyl-3-hydroxyphthalimide (BHIO), an acid-binding agent and a catalyst 4-Dimethylaminopyridine (DMAP) in dichloromethane, and reacting at 0-30 ℃;
2) dissolving pyridine-2-formyl chloride hydrochloride in dichloromethane, slowly dropwise adding the dichloromethane into a reaction solution, and purifying after the reaction is finished to obtain the phthalimide fluorescent probe for detecting copper ions.
According to the technical scheme, N-butyl-3-hydroxyphthalimide and pyridine-2-formyl chloride are used as raw materials, and the fluorescent probe is prepared through acylation reaction under the catalysis of alkali and 4-dimethylaminopyridine. The method can prepare the fluorescent probe LXJ-3 through two steps, and the synthesis process is simple.
Specifically, a compound BHIO, an acid-binding agent and DMAP are dissolved in dichloromethane, pyridine-2-formyl chloride hydrochloride is dissolved in dichloromethane, the mixture is dripped into a reaction liquid through a constant-pressure dropping funnel under stirring, reaction is carried out at 0-30 ℃, and the phthalimide probe LXJ-3 is obtained through purification after the reaction is finished.
The compound BHIO of the invention is prepared by heating 3-hydroxyphthalic anhydride and n-butylamine in acetic acid by a disclosed method (analysis laboratory 2020, 39(12): 1430-1434).
The chemical name of compound BHIO is N-butyl-3-hydroxyphthalimide, and the structural formula is as follows:
specifically, in the step 2), the reaction time is 4-10 h.
Specifically, in the step 1), the acid-binding agent is selected from one or more of N, N-Diisopropylethylamine (DIPEA), triethylamine or pyridine.
Specifically, in the step 2), the reaction solution obtained after the reaction is finished is subjected to reduced pressure distillation to remove the solvent, so as to obtain a crude product, and then the crude product is separated and purified by a silica gel column, wherein the mobile phase is a mixed solvent of ethyl acetate and petroleum ether in a volume ratio of 1: 2.
Specifically, the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the pyridine-2-formyl chloride hydrochloride is 1 (1-2); the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the acid-binding agent is 1 (2-4); the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the catalyst is (5-20): 1.
The invention also provides application of the phthalimide fluorescent probe for detecting copper ions, and the phthalimide fluorescent probe is used as Cu2+Fluorescent probe of (2), qualitative or quantitative detection of Cu2+The concentration of (c).
The technical proposal utilizes Cu2+And catalyzing the probe LXJ-3 in a buffer solution to perform hydrolysis reaction, removing pyridine-2-formyl and regenerating a fluorescence parent BHIO, so that the ESIPT process is recovered, and the fluorescence is obviously enhanced. Specifically, under the irradiation of 365nm ultraviolet lamp, the probe solution has weak fluorescence, and Cu is added2+Then strong green fluorescence is emitted, thereby realizing the aim of Cu2+Detection of (3). Probe LXJ-3 vs. Cu in PBS buffer (10mM, pH 7.4, physiological conditions pH 7.4 being chosen for ease of use in biology)2+Has specific selectivity, strong anti-interference capability and obvious fluorescence enhancement effect, and the detection limit is 31 nM.
Specifically, Cu2+The detection method comprises the following steps:
1) establishing a standard curve of the gradient change of the concentration of the copper ions and the change value of the fluorescence intensity of the fluorescent probe solution;
2) obtaining Cu in the solution to be detected from the standard curve according to the fluorescence intensity of the solution to be detected2+The concentration of the ions.
More specifically, the method comprises the following steps:
1) respectively mixing the copper ion solution with the gradient change of the concentration with the probe LXJ-3 solution with the specific concentration, measuring the corresponding fluorescence intensity, then drawing by taking the concentration of the copper ions as an abscissa and the fluorescence intensity of the corresponding mixed solution as an ordinate, and establishing a standard curve of the gradient change of the concentration of the copper ions and the change value of the fluorescence intensity of the probe LXJ-3 solution;
2) and obtaining the concentration of the copper ions in the solution to be detected from the standard curve according to the fluorescence intensity.
Specifically, the fluorescence detection conditions are as follows: the excitation wavelength is 344nm, the maximum fluorescence emission wavelength is 515nm, the slit width is 5nm and 5nm, and the fluorescence emission spectrum is detected at 420-650 nm.
Specifically, the fluorescent probe stock solution adopted by the fluorescence detection comprises a fluorescent probe, a solvent and a buffer solution, wherein the fluorescent probe is phthalimide derivative LXJ-3.
Specifically, the molar concentration of the fluorescent probe in the stock solution is 0.01-10 mM; the solvent is at least one of methanol, ethanol and dimethyl sulfoxide; the buffer solution is PBS buffer solution or Tris-HCl buffer solution, and the pH value of the buffer solution is 5-10.
Compared with the prior art, the invention has the following advantages:
the phthalimide fluorescent probe LXJ-3 provided by the invention has the advantages of simple structure, easy synthesis and higher yield. Probe LXJ-3 realizes selective recognition of copper ions based on ESIPT process of N-butyl-3-hydroxyphthalimide, has the advantages of large Stokes displacement, high fluorescence quantum yield, strong optical stability and the like, and can sensitively and specifically recognize and detect Cu in a pure water phase and a wider pH range2+The method is not interfered by other ions basically, the minimum detection limit of the method to the copper ions is 31nM, the quantitative detection of trace copper ions in complex environment or in organisms can be well met, and the method has important application value.
Drawings
FIG. 1 is a view of probe LXJ-3 prepared in example 11H-NMR spectrum;
FIG. 2 is a view of probe LXJ-3 prepared in example 113A C-NMR spectrum;
FIG. 3 is a high resolution mass spectrum of probe LXJ-3 prepared in example 1;
FIG. 4 is a graph showing the change of fluorescence intensity with time of the reaction of probe LXJ-3 prepared in example 1 with copper ions;
FIG. 5 is a graph showing the change in fluorescence spectrum of probe LXJ-3 prepared in example 1 when copper ions were added at different concentrations;
FIG. 6 is a graph showing the change in fluorescence intensity of probe LXJ-3 prepared in example 1 when added to copper ions of different concentrations and the linear graph showing the fluorescence intensity versus the concentration of copper ions;
FIG. 7 is a graph showing the change of fluorescence response with pH of probe LXJ-3 and probe LXJ-3 reacted with copper ions prepared in example 1;
FIG. 8 shows fluorescence spectra of probe LXJ-3 prepared in example 1 with different interfering ions added in PBS buffer system;
FIG. 9 shows the effect of other interfering ions on the fluorescence intensity of probe LXJ-3 plus copper ion system prepared in example 1;
FIG. 10 shows pairs of probes LXJ-3 and Cu prepared in example 12+A possible recognition mechanism;
FIG. 11 shows the presence and absence of Cu2+Fluorescence spectra of probe LXJ-3 (10. mu.M) prepared in example 1 and BHIO (10. mu.M) in PBS (10mM, pH 7.4) (20. mu.M);
FIG. 12 shows probe LXJ-3 and Cu prepared in example 12+High resolution mass spectrogram of the reacted solution.
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
The following discloses many different embodiments or examples for implementing the subject technology described. While specific examples of one or more arrangements of features are described below to simplify the disclosure, the examples should not be construed as limiting the invention, and the first feature described later in the specification in conjunction with the second feature may include embodiments that are directly related, may also include embodiments that form additional features, and further may include embodiments in which one or more additional intervening features are used to indirectly connect or combine the first and second features to each other, so that the first and second features may not be directly related.
In embodiments disclosed herein are phthalimide derivatives having the formula:
the application also discloses a preparation method of the phthalimide derivative, and the synthetic route is as follows:
for ease of understanding, the present application provides several specific implementations:
example 1
The phthalimide derivative is prepared by the following steps, and specifically comprises the following steps:
BHIO (0.1g,0.46mmol), DIPEA (0.18g,1.38 mmol), DMAP (6mg,0.046mmol) and 15mL of dried dichloromethane are added into a 100mL round-bottom flask, stirring is carried out at room temperature, pyridine-2-formyl chloride hydrochloride (0.12g,0.69mmol) is dissolved in 5mL of dried dichloromethane and slowly dropped into the reaction liquid, after the reaction is completed for 8 hours by dropping, TLC monitors that the reaction is completed, and the solvent is removed by distillation under reduced pressure to obtain a crude product; passing through silica gel column (mobile phase is petroleum ether: ethyl acetate: 2:1, V: V) to obtain white solid product 0.13g, yield 85%.
The nuclear magnetic analysis of the obtained product showed the following results:
1H NMR(400MHz,DMSO-d6)δ:8.84-8.86(m,1H),8.28-8.30(m,1H), 8.10-8.15(m,1H),7.95(t,1H,J=7.5Hz),7.84(d,1H,J=7.4Hz),7.75-7.80(m, 2H),3.50(t,2H,J=7.0Hz),1.48-1.55(m,2H),1.20-1.29(m,2H),0.85(t,3H, J=7.4Hz);13C NMR(100MHz,DMSO-d6)δ:167.02,165.53,162.54,150.21, 146.02,145.96,137.87,136.56,133.27,128.73,128.31,126.03,122.34,121.13, 37.20,29.85,19.46,13.43.
calculated value of ESI-HRMS (m/z) C18H16N2NaO4[M+Na]+347.10023, found 347.10040.
Example 2
BHIO (0.1g,0.46mmol), DIPEA (0.24g, 1.84mmol), DMAP (12mg,0.092mmol) and 15mL of dried dichloromethane are added into a 100mL round-bottom flask, stirring is carried out at room temperature, pyridine-2-formyl chloride hydrochloride (0.16g,0.92mmol) is dissolved in 5mL of dried dichloromethane and slowly dropped into the reaction liquid, after 4 hours of reaction is completed, TLC monitors that the reaction is completed, and the solvent is removed by distillation under reduced pressure to obtain a crude product; passing through silica gel column (mobile phase is petroleum ether: ethyl acetate: 2:1, V: V) to obtain white solid product 0.13g, yield 88%.
Example 3
BHIO (0.1g,0.46mmol), triethylamine (0.14g,1.38 mmol), DMAP (6mg,0.046mmol) and 15mL of dried dichloromethane are added into a 100mL round-bottom flask, stirring is carried out at room temperature, pyridine-2-formyl chloride hydrochloride (0.12g,0.69mmol) is dissolved in 5mL of dried dichloromethane and slowly dropped into the reaction liquid, after the completion of the reaction for 8 hours, TLC monitors the completion of the reaction, and the solvent is removed by distillation under reduced pressure to obtain a crude product; passing through silica gel column (mobile phase is petroleum ether: ethyl acetate: 2:1, V: V) to obtain white solid product 0.12g, yield 80%.
Example 4
BHIO (0.1g,0.46mmol), pyridine (0.11g,1.38 mmol), DMAP (6mg,0.046mmol) and 15mL of dried dichloromethane are added into a 100mL round-bottom flask, stirring is carried out at room temperature, pyridine-2-formyl chloride hydrochloride (0.123g,0.69mmol) is dissolved in 5mL of dried dichloromethane and slowly dropped into the reaction liquid, after 8 hours of dropping reaction, TLC monitors that the reaction is complete, and the solvent is removed by distillation under reduced pressure to obtain a crude product; passing through silica gel column (mobile phase is petroleum ether: ethyl acetate: 2:1, V: V) to obtain white solid product 0.11g, yield 76%.
The application also discloses several embodiments of the phthalimide derivative used as a fluorescent probe for detecting copper ions.
Example 5
Method for testing spectral properties
Dissolving the probe LXJ-3 in DMSO to prepare a 5mM stock solution, and storing at low temperature; metal cation (K)+、Na+、Ag+、Ca2+、Mg2+、Cu2+、Zn2+、Al3+、Fe3+、Fe2+、Cd2+、Cr3+、 Co2+、Hg2+、Ni2+、Pb2+、Mn2+) Dissolving with secondary water to obtain a solution with the concentration of 10 mM. The determination system comprises: mu.L of the stock solution of the probe LXJ-3 and 30. mu.L of the solution of the metal ion to be detected were mixed, and the mixture was made to volume of 3mL with PBS buffer solution, placed in a cuvette, reacted for 15min, and then measured. And (3) testing conditions are as follows: room temperature, the parameter of the fluorescence spectrophotometer is lambdaex=344nm,λem515nm, slit widths of 5nm and 5nm, voltage of 700V, wavelength range of 420 and 650 nm.
Example 6
Probe LXJ-3 and Cu2+Influence of reaction time on fluorescence intensity
In the PBS buffer solution (10mM, pH 7.4) system, the fluorescence of the probe LXJ-3(10 μ M) solution itself is very weak; 2 equivalents of Cu were added to the probe LXJ-3 solution2+Then, the fluorescence intensity of the probe is recorded at regular intervals, and the result shows that the fluorescence intensity at 515nm is rapidly enhanced, the increase trend of the fluorescence intensity gradually becomes smaller along with the progress of the reaction, and the fluorescence intensity basically tends to be stable after 15 min. Therefore, the reaction time was controlled within 15min in all subsequent experiments (FIG. 4).
Example 7
Cu2+Influence of concentration of (2) on fluorescence intensity of Probe LXJ-3
As shown in FIG. 5, Cu was contained in PBS buffer (10mM, pH 7.4)2+The fluorescence intensity of probe LXJ-3(10 μ M) at 515nm is dependent on Cu in the concentration range of 0-50 μ M2+The increase in concentration is markedly enhanced. As can be seen from FIG. 6, in Cu2+When the concentration was increased to 20. mu.M, the fluorescence intensity of probe LXJ-3 reached a maximum, and further increase of Cu was continued2+The fluorescence intensity remains substantially unchanged. The fluorescence intensity of the probe LXJ-3 and Cu in the concentration range of 0-10 μ M2+Has a good linear relation with the regression equation of y being 532.9x +909.7 (R)20.9938). Calculating a formula according to the limit of detection (LOD): LOD is 3 sigma/K (wherein sigma is Cu is not added)2+Standard deviation of fluorescence spectrum change of the time probe LXJ-3, and K is the slope of a straight line), calculating to obtain the probe LXJ-3 for Cu2+The detection limit of (2) was 31nM, indicating that probe LXJ-3 is paired with Cu2+Has better detection sensitivity.
Example 8
Effect of pH on the detection Performance of Probe LXJ-3
As shown in FIG. 7, in the PBS buffer solution (10mM, pH 7.4) system, the fluorescence intensity of the probe LXJ-3(10 μ M) solution does not change much in the range of pH 3-11, indicating that the probe LXJ-3 is relatively stable under acidic and weakly alkaline conditions. When 2 equivalents of Cu are added2+Then, the fluorescence intensity of the probe LXJ-3 and the fluorescence intensity of the probe without Cu are within the pH range of 3-42+The time variation is not large; and in the range of pH 5-10, probes LXJ-3 and Cu2+The increase in fluorescence intensity after the action was very significant and reached a peak at pH 7.4. Indicating that probe LXJ-3 can detect copper ions over a wide pH range. To facilitate its use in biology, the PBS buffer system at physiological conditions pH 7.4 was chosen for testing.
Example 9
Selectivity and interference of probe LXJ-3 with other metal ions
Selection of cations (K) which may cause interference+、Na+、Ag+、Ca2+、Mg2+、Zn2+、Al3+、 Fe3+、Fe2+、Cd2+、Cr3 +、Co2+、Hg2+、Ni2+、Pb2+、Mn2+) To examine probe LXJ-3 for Cu2+Selectivity of (2). The change in fluorescence spectrum of probe LXJ-3 was measured after reaction at room temperature for 15min by adding 2 equivalents of each of the above ions to 10. mu. mol/L of probe LXJ-3 in PBS buffer (10mM, pH 7.4). When interfering ions are added, the fluorescence of probe LXJ-3 is not significantly changed; while when 2 equivalents of Cu are added2+After that, the fluorescence intensity was significantly increased (fig. 8). Thus, probe LXJ-3 was aligned with Cu2+Has specific selectivity and can be used for Cu2+The analysis and detection of (2).
To further test probes LXJ-3 in Cu2+Detection of Cu in the Presence of other competitive ions2+For probe LXJ-3, an anti-interference experiment was performed. 10 μ M Probe LXJ-3 and 2 equivalents Cu in PBS buffer (10mM, pH 7.4) system2+After mixing, 2 equivalents of each of the above ionic solutions was added, and the change in fluorescence intensity of the mixed solution was measured. As shown in FIG. 9, the fluorescence intensity of probe LXJ-3 was compared with that of Cu alone2+When present, are substantially the same. The above results indicate that the presence of other competitive materials detects Cu for probe LXJ-32+No significant interference is generated. Thus, probe LXJ-3 can be used for Cu in complex environments2+Detection of (3).
Example 10
Probe LXJ-3 pairs of Cu2+Mechanism of recognition of
Identification of Cu by pyridine-2-formyl group was reported according to the literature (sensor. actuat. b-chem.,2017,252,134.)2+We propose LXJ-3 pairs of Cu2+Possible response mechanisms (fig. 10). Because of the presence of pyridine-2-formyl, the ESIPT process based on keto-enol tautomer in BHIO was inhibited, and probe LXJ-3 itself exhibited weak fluorescence. When the probe and Cu are present2+When the probe is used, pyridine-2-formate is catalyzed and hydrolyzed, the probe LXJ-3 removes pyridine-2-formyl, the fluorescent parent BHIO is released to recover the ESIPT process, and strong green fluorescence is re-presented, so that the probe is prepared by the methodRealization of Cu2+Identification and detection. To verify our conjecture, LXJ-3 and Cu were paired2+The fluorescence spectrum and mass spectrum of the product after the reaction were analyzed. Fluorescence spectrum experiments show that under excitation of 344nm, the fluorescence of the probe LXJ-3 is very weak, while when LXJ-3 and Cu are used2+After the reaction, a significantly enhanced fluorescence signal appeared at 515nm, which is consistent with the fluorescence spectrum of the parent fluorescent BHIO (see FIG. 11). LXJ-3 and Cu2+An ion peak at M/z 218.08215 appears in the high resolution mass spectrum after the reaction (see FIG. 12), which is related to the deprotonated molecular weight of the parent BHIO [ M-H ]]-218.08227, in accordance.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (9)
2. the method for preparing the phthalimide-based fluorescent probe for detecting copper ions according to claim 1, comprising the steps of:
1) dissolving N-butyl-3-hydroxyphthalimide, an acid-binding agent and 4-dimethylaminopyridine serving as a catalyst in dichloromethane, and reacting at 0-30 ℃;
2) dissolving pyridine-2-formyl chloride hydrochloride in dichloromethane, slowly dropwise adding the dichloromethane into a reaction solution, and purifying after the reaction is finished to obtain the phthalimide fluorescent probe for detecting copper ions.
3. The method for preparing a phthalimide-based fluorescent probe for detecting copper ions according to claim 2, wherein the method comprises: in the step 2), the reaction time is 4-10 h.
4. The method for preparing a phthalimide-based fluorescent probe for detecting copper ions according to claim 2, wherein the method comprises: in the step 1), the acid-binding agent is selected from one or more of N, N-Diisopropylethylamine (DIPEA), triethylamine or pyridine.
5. The method for preparing a phthalimide-based fluorescent probe for detecting copper ions according to claim 2, wherein the method comprises: in the step 2), the purification specifically comprises the steps of distilling the reaction liquid obtained after the reaction under reduced pressure to remove the solvent to obtain a crude product, and then separating and purifying the crude product through a silica gel column, wherein the mobile phase is a mixed solvent of ethyl acetate and petroleum ether in a volume ratio of 1: 2.
6. The method for producing a phthalimide-based fluorescent probe for detecting copper ions according to any one of claims 2 to 5, wherein: the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the pyridine-2-formyl chloride hydrochloride is 1 (1-2); the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the acid-binding agent is 1 (2-4); the molar ratio of the compound N-butyl-3-hydroxyphthalimide to the catalyst is (5-20): 1.
7. Use of a phthalimide-based fluorescent probe for detecting copper ions according to claim 1, wherein: as Cu2+The fluorescent probe of (1).
8. A method for detecting copper ions is characterized by comprising the following steps:
1) establishing a standard curve of the change of the copper ion concentration gradient and the change value of the fluorescence intensity of the phthalimide fluorescent probe solution for detecting copper ions according to claim 1;
2) detecting fluorescence of a test solutionThe light intensity is measured, and Cu in the solution to be measured is obtained from the standard curve according to the fluorescence intensity of the solution to be measured2+The concentration of the ions.
9. The method for detecting copper ions according to claim 8, wherein the fluorescence detection conditions in each step are as follows: the excitation wavelength is 344nm, the maximum fluorescence emission wavelength is 515nm, the slit width is 5nm and 5nm, and the fluorescence emission spectrum is detected at 420-650 nm.
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