A fluorescence-enhanced copper ion probe and its preparation method and application

Abstract

The invention discloses a fluorescence-enhanced copper ion probe and preparation method and application. The method includes: 5,10-diformylphenyl-15,20-disulfonic acid phenyl metalloporphyrin (cis-MFTPPS), 2-hydrazine pyridine are dissolved in N, N-dimethylformamide with a molar ratio of 1:3, under nitrogen protection, heated to reflux, reacted 8-12h, after cooling, decompression distillation to remove the solvent, after reverse chromatographic column separation, in a 1000kDa dialysis bag, dialyzed for 48-55 hours to obtain a target copper ion fluorescent probe (MP). The probe preparation method is simple, the conditions are mild, and the post-processing is simple. The signal-enhanced fluorescent response to copper ions can be achieved in 100% aqueous solution, and the detection sensitivity is high, and the detection process is convenient.

Description

Technical Field

[0001] The present invention relates to the preparation of a fluorescence-enhanced probe for detecting copper ions, and more particularly to a method for preparing a water-soluble copper ion fluorescent probe with a nitrogen-nitrogen atom cavity as a recognition group and a metalloporphyrin molecule as a fluorescent chromophore, and the application of the probe molecule in the fluorescence detection of copper ions in aqueous solution. Background Art

[0002] With the rapid development of modern industry, heavy metal ion pollution is becoming more and more serious. 2+ ) is widely used in industry, medicine, agriculture and other fields, Cu 2+ It is considered to be one of the most important environmental and ecological pollutants. Copper is very toxic to aquatic organisms. 2+ Wastewater irrigation farmland, Cu 2+ Cu can accumulate in soil and crops, causing poor growth of crops, especially rice and barley, and contaminating grains. 2 + , through the food chain, a large amount of Cu 2+ Entering the human body, it causes metabolic disorders in human cells and leads to various neurological diseases, such as Wilson's disease, Alzheimer's disease and Menke's disease. Therefore, people urgently need to analyze and detect Cu in water quickly, accurately, at low cost and selectively. 2+ Develop an efficient and simple method for the detection of trace Cu in environmental samples. 2 + A detection method is imperative.

[0003] Among various detection methods, fluorescence technology has become the most popular method for Cu detection due to its high sensitivity, strong specificity, simple operation, real-time monitoring and fast response time. 2+ The most commonly used method for detection. For example, the patent with publication number CN105223174A, “A detection method based on fluorescence quenching of copper ions”, and the patent with publication number CN104961647A, “Fluorescence recognition of copper ion sensor molecules and their synthesis and application”, both reported different types of copper ion fluorescent probes. Although these probes have high selectivity and sensitivity to copper ions, however, most of the reported copper ion fluorescent probes are sensitive to Cu. 2+It shows a fluorescence quenching response. This response mode has a lower sensitivity than the fluorescence enhanced probe. Although some patents, such as the patent with publication number CN109942509A "A fluorescent probe for identifying copper ions under alkaline conditions, its preparation method and application", the patent with publication number CN103013495A "A copper ion fluorescent probe and its synthesis method", and the patent with publication number CN108250188A "A long wavelength fluorescent probe for detecting copper ions, its synthesis method and application", have reported fluorescence enhanced Cu 2+ Fluorescent probes. However, these probes have complicated synthesis steps, low yields, or poor water solubility and cannot be detected in pure aqueous solutions, which greatly limits their further application. Therefore, improving the water solubility of the probe molecules and constructing fluorescent signal-enhanced Cu 2+ Fluorescent probes are the key to solving the above problems. Summary of the Invention

[0004] In response to the problems of existing fluorescent probes such as complex preparation, poor water solubility and insignificant fluorescence signal changes, the present invention provides a water-soluble fluorescence-enhanced copper ion fluorescent probe with a nitrogen-nitrogen atom cavity as a recognition group and a metal porphyrin molecule as a fluorescent chromophore, and a preparation method thereof.

[0005] The fluorescence-enhanced copper ion fluorescent probe (MP) provided by the present invention has the following general structural formula:

[0006]

[0007] Where M = Zn 2+ or Mg 2+ .

[0008] The method for preparing the fluorescence-enhanced copper ion fluorescent probe provided by the present invention adopts the following technical scheme:

[0009] A method for preparing a fluorescence-enhanced copper ion fluorescent probe (MP) is characterized by comprising the following steps: dissolving 5,10-diformylphenyl-15,20-disulfonic acid phenyl metalloporphyrin (cis-MFTPPS) and 2-hydrazinepyridine in an organic solvent at a molar ratio suitable for the reaction, heating to reflux under nitrogen protection, reacting for 8 to 12 hours, cooling, removing the solvent by reduced pressure distillation, separating by reverse phase chromatography, and dialyzing in a 1000 kDa dialysis bag to obtain the target copper ion fluorescent probe (MP).

[0010] The molar ratio suitable for the reaction is preferably 1:3 of 5,10-diformylphenyl-15,20-disulfonic acid phenyl metalloporphyrin (cis-MFTPPS):2-hydrazinepyridine.

[0011] The preparation method of the fluorescence-enhanced copper ion fluorescent probe (MP) specifically comprises the following steps:

[0012] (1) Paraformaldehyde and sodium chloride were mixed in a mass ratio of 6:1 and dissolved in dichloromethane to form a 30 mg / mL paraformaldehyde solution. Chlorosulfonic acid (the mass ratio of chlorosulfonic acid to paraformaldehyde was 6:1) was added to the mixture under ice-salt bath (-10°C). After vigorous stirring for 20-30 min, commercially available tetraphenylporphyrin (TPP) was dissolved in dichloromethane to form a 50 mg / mL tetraphenylporphyrin (TPP) solution. The solution was slowly added dropwise to a round-bottom flask at a rate of 3 min / mL. The mass ratio of paraformaldehyde to tetraphenylporphyrin (TPP) in the reaction solution was 7.5:1. The reaction was monitored by thin layer chromatography (TLC) until no tetraphenylporphyrin (TPP) raw material remained. The reaction was then stopped and the crude product was separated by silica gel chromatography. The second color band product was collected. 5,10-dichloromethylphenyl-15,20-diphenylporphyrin (cis-ClTPP) was obtained.

[0013] The structural formula of cis-ClTPP is:

[0014]

[0015] (2) 5,10-dichloromethylphenyl-15,20-diphenylporphyrin (cis-ClTPP) was dissolved in chloroform to form a solution with a concentration of 3 mg / mL, and hexamethylenetetramine was added (the mass ratio of cis-ClTPP to hexamethylenetetramine was 1:8). The reaction was carried out at 45°C. After thin layer chromatography (TLC) was used to verify that no cis-ClTPP raw material remained, the reaction was stopped. The solvent was removed by rotary evaporation, and 60% acetic acid aqueous solution was added. After reflux at 100-120°C for 8-12 hours, the solution was cooled, and a large amount of water was added to precipitate a solid. The solid was washed with water until neutral, thereby obtaining 5,10-diformylphenyl-15,20-diphenylporphyrin (cis-FTPP).

[0016] The structural formula of cis-FTPP is:

[0017]

[0018] (3) 5,10-diformylphenyl-15,20-diphenylporphyrin (cis-FTPP) was dissolved in concentrated sulfuric acid to form a solution with a concentration of 10 mg / mL, and the solution was heated to reflux and reacted for 6 to 9 hours. The degree of reaction was determined by thin layer chromatography (TLC). After the FTPP was completely reacted, the heating was stopped. The reaction solution was neutralized by adding 1 mol / L sodium hydroxide solution to a pH of 7, and dialyzed in a 1000 kDa dialysis bag for 45 to 50 hours. The solution was freeze-dried for 20 to 28 hours to obtain water-soluble 5,10-diformylphenyl-15,20-disulfonic acid phenylporphyrin (cis-FTPPS).

[0019] The structural formula of cis-FTPPS is:

[0020]

[0021] (4) 5,10-diformylphenyl-15,20-disulfonic acid phenylporphyrin (cis-FTPPS) and metal acetate were mixed at a molar ratio of 1:2 and dissolved in an organic solvent to form a solution with a cis-FTPPS concentration of 0.005 mmol / mL; after refluxing at 153°C for 4-5 hours, the organic solvent was removed by vacuum distillation, and the solution was dialyzed in a 1000 kDa dialysis bag for 48-72 hours and freeze-dried for 20-28 hours to obtain 5,10-diformylphenyl-15,20-disulfonic acid phenyl metalloporphyrin (cis-MFTPPS).

[0022] The structural formula of cis-MFTPPS is:

[0023]

[0024] Where M = Zn 2+ or Mg 2+

[0025] (5) 5,10-diformylphenyl-15,20-disulfonate phenyl metalloporphyrin (cis-MFTPPS) and 2-hydrazinepyridine were dissolved in an organic solvent at a molar ratio of 1:3. The volume of the organic solvent was proportional to the amount of 5,10-diformylphenyl-15,20-disulfonate phenyl metalloporphyrin (cis-MFTPPS), and the concentration of 5,10-diformylphenyl-15,20-disulfonate phenyl metalloporphyrin (cis-MFTPPS) in the solution was maintained at 0.025 mmol / mL. After heating under reflux (153°C) for 8-12 h, the reaction was cooled and the solvent was removed by vacuum distillation. After separation by reverse phase chromatography, the target copper ion fluorescent probe (MP) was obtained after 48-55 h of dialysis in a 1000 kDa dialysis bag.

[0026] In the step (1), the optimal dropwise addition rate of tetraphenylporphyrin (TPP) is 3 min / mL. This rate allows TPP to evenly enter the solution, making it difficult for the product to aggregate, which is conducive to the full progress of the reaction.

[0027] The preferred mass ratio of paraformaldehyde to tetraphenylporphyrin (TPP) in step (1) is 7.5:1. This ratio will result in a higher amount of 5,10-dichloromethylphenyl-15,20-diphenylporphyrin (cis-ClTPP) produced by the reaction and a lower amount of by-products such as monosubstituted chloromethylporphyrin.

[0028] In the step (1), the developing solvent for silica gel chromatography separation is dichloromethane: petroleum ether = 1:1 (volume ratio); in the step (2), the developing solvent for silica gel chromatography separation is dichloromethane: petroleum ether = 2:1 (volume ratio); in the step (3), the developing solvent for silica gel chromatography separation is dichloromethane: methanol = 3:1 (volume ratio).

[0029] In the step (2), the mass ratio of 5,10-dichloromethylphenyl-15,20-diphenylporphyrin (cis-ClTPP) to hexamethylenetetramine is preferably 1:8, and the optimal concentration of the acetic acid solution is 60%. These two ratios can fully oxidize and reduce the chloromethyl groups on 5,10-dichloromethylphenyl-15,20-diphenylporphyrin (cis-ClTPP) to form formyl groups, which is more conducive to the full progress of the reaction.

[0030] In the step (4), the metal acetate is magnesium acetate tetrahydrate or zinc acetate dihydrate.

[0031] The organic solvent in step (4) and step (5) is N,N-dimethylformamide (DMF). In step (4), the molar ratio of 5,10-diformylphenyl-15,20-disulfonatephenylporphyrin (cis-FTPPS) to metal acetate is selected to be 1:2, which can ensure that cis-FTPPS is converted into cis-MFTPPS with the highest efficiency while minimizing the use of materials, thereby facilitating the efficient reaction.

[0032] In the step (5), the molar ratio of cis-MFTPPS to 2-hydrazinepyridine is preferably 1:3. Experiments have shown that this ratio can ensure the highest utilization of the reaction substrate, the shortest required reaction time, and the least by-products.

[0033] The volume of the organic solvent in step (5) is proportional to the amount of 5,10-diformylphenyl-15,20-disulfonic acid phenyl metalloporphyrin (cis-MFTPPS), and the concentration of 5,10-diformylphenyl-15,20-disulfonic acid phenyl metalloporphyrin (cis-MFTPPS) in the solution is maintained at 0.025 mmol / mL. This ratio allows the reaction raw materials to be most fully dissolved with the least amount of solvent used, which is more conducive to the full progress of the reaction.

[0034] In the step (5), the reverse chromatography eluent is a methanol-water system, and the elution gradient ranges from 0% to 85% (volume ratio) of methanol.

[0035] The fluorescence-enhanced copper ion fluorescent probe (MP) can undergo a unique coordination reaction with copper ions as shown in the following formula.

[0036]

[0037] Where M = Zn 2+ or Mg 2+ .

[0038] The fluorescence enhanced copper ion fluorescent probe (MP) is not 2+ When complexed, the nitrogen atom on the imine group can undergo intramolecular photoinduced electron transfer (PET) with the porphyrin ring, causing fluorescence quenching of the MP molecule; when combined with Cu 2+ After coordination, the original PET effect is blocked, a more rigid molecular plane is formed, and the π-electron conjugated system of the molecule is expanded, resulting in enhanced fluorescence signals.

[0039] The present invention has the following advantages:

[0040] (1) The synthesis method of the copper ion fluorescent probe (MP) of the present invention has mild conditions, simple steps, and simple post-processing;

[0041] (2) The present invention uses porphyrin molecules as signal reporting groups. These molecules are near-infrared fluorescent chromophore molecules with excellent optical properties, are sensitive to the microenvironment, and can obtain a large Stokes shift, thereby enhancing the sensitivity of the reaction signal.

[0042] (3) The copper ion fluorescent probe (MP) of the present invention can show a fluorescence-enhanced signal response to copper ions and has high sensitivity;

[0043] (4) The copper ion fluorescent probe (MP) of the present invention can be used in 100% aqueous solution without the need for adding additional organic solvents, which makes the detection range wider and the detection results more accurate.

[0044] (5) The nitrogen-nitrogen atom cavity of the recognition group in the copper ion fluorescent probe (MP) of the present invention is Cu 2+ The probe has high thermodynamic affinity and fast metal-ligand binding kinetics, making it suitable for Cu 2+ It has strong specificity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The MP1 probe prepared in Example 1 of the present invention 1 H NMR spectrum, where 1 H NMR (500 MHz, DMSO-d6):δ H ,ppm 9.9(s,1H),9.76(s,1H),8.77-8.98(m,10H), 8.65-8.71(m,2H),8.18-8.24(m,8H),8.06-8.15(m,8H),7.45-7.48(m,6H).

[0046] Figure 2 The ultraviolet and fluorescence spectra of the MP1 probe prepared in Example 1 of the present invention;

[0047] Figure 3 This is the copper ion titration fluorescence spectrum of the MP1 probe prepared in Example 1 of the present invention;

[0048] Figure 4 Fluorescence photos of the MP1 probe prepared in Example 1 of the present invention before and after the interaction with copper ions (under ultraviolet light, excitation wavelength: 365 nm);

[0049] Figure 5 This is a graph showing the fluorescence changes of the MP1 probe solution prepared in Example 1 of the present invention to different metal ions. DETAILED DESCRIPTION

[0050] Example 1

[0051] 600 mg of paraformaldehyde and 100 mg of sodium chloride were placed in a 100 mL round-bottom flask, followed by 20 mL of dichloromethane. 2.05 mL of chlorosulfonic acid was added under ice-salt bath (-10°C) with vigorous stirring to dissolve the mixture. After 20 min of dissolution, tetraphenylporphyrin (TPP) (80 mg) was dissolved in 1.6 mL of dichloromethane and slowly added dropwise to the round-bottom flask at a constant rate over 5 min. The reaction progress was monitored by thin-layer chromatography (TLC) (developing solvent: dichloromethane: petroleum ether = 1:1 (v / v)). The reaction was terminated when no TPP was present. The solution was washed with water several times until pH = 7. The organic phase was collected, the solvent was rotary evaporated, and the crude product was separated by silica gel chromatography (developing solvent: dichloromethane: petroleum ether = 1:1 (v / v)). The second band of product was collected to obtain 5,10-dichloromethylphenyl-15,20-diphenylporphyrin (cis-ClTPP). Using nuclear magnetic resonance spectroscopy ( 1 H NMR) confirmed its chemical structure. 1 Deuterated chloroform (CDCl 3 ) was used as the solvent for H NMR.

[0052] 5,10-Dichloromethylphenyl-15,20-diphenylporphyrin (cis-ClTPP) (60 mg, 0.0846 mmol) was dissolved in 120 mL of chloroform. 480 mg of hexamethylenetetramine was added and the reaction was allowed to proceed at 45°C. The reaction progress was monitored by thin-layer chromatography (TLC) (developing solvent: dichloromethane:petroleum ether = 2:1 (v / v)). The reaction was stopped when no cis-ClTPP was present. The solvent was removed by rotary evaporation, and 20 mL of 60% aqueous acetic acid was added. The reaction was continued at 110°C for 8 h. After the reaction, the solution was cooled and 100 mL of water was added. A dark red solid precipitated, which was filtered and washed with water until the filtrate was neutral. The filter cake was collected, dissolved in chloroform, dried over anhydrous sodium sulfate overnight, and the solvent was evaporated to obtain 5,10-diformylphenyl-15,20-diphenylporphyrin (cis-FTPP). Using nuclear magnetic resonance spectroscopy ( 1 H NMR) confirmed its chemical structure. 1 Deuterated chloroform (CDCl 3 ) was used as the solvent for H NMR.

[0053] 5,10-diformylphenyl-15,20-diphenylporphyrin (cis-FTPP) (40 mg, 0.06 mmol) was dissolved in 4 mL of concentrated sulfuric acid, heated to reflux, and the reaction was stopped after 8 hours. The reaction solution was neutralized with 1 mol / L sodium hydroxide solution to a pH of 7, dialyzed in a 1000 kDa dialysis bag for 48 hours, and freeze-dried for 26 hours to obtain 5,10-diformylphenyl-15,20-disulfonic acid phenyl metalloporphyrin (cis-FTPPS).

[0054] 5,10-Diformylphenyl-15,20-disulfonate phenyl metalloporphyrin (cis-FTPPS) (100 mg, 0.114 mmol) was dissolved in 22.8 mL of DMF, and 0.4 mL of triethylamine and zinc acetate dihydrate (50 mg, 0.228 mmol) were added. The mixture was refluxed at 153°C for 4 h. After completion of the reaction, the mixture was cooled to room temperature, and the solvent was removed by distillation under reduced pressure. The mixture was dialyzed in a 1000 kDa dialysis bag for 50 h and freeze-dried for 22 h to obtain 5,10-diformylphenyl-15,20-disulfonate phenyl zinc porphyrin (cis-ZnFTPPS).

[0055] 5,10-diformylphenyl-15,20-disulfonic acid phenyl zinc porphyrin (cis-ZnFTPPS) (100 mg, 0.107 mmol) was dissolved in 4.3 mL of DMF. After dissolution, 2-hydrazinepyridine (35.0 mg, 0.321 mmol) was added and refluxed at 153°C for 9 hours. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed by distillation under reduced pressure. Reverse column chromatography was used for separation. The eluent was a methanol-water system with an elution gradient of methanol from 0% to 70% (volume ratio) to obtain the final copper ion fluorescent probe (MP1). The weight was 89.3 mg and the yield was 74.6%. The H NMR spectrum ( 1 (H NMR) Figure 1 ) and UV fluorescence spectra ( Figure 2 ) to verify its chemical structure. 1 Deuterated DMSO (DMSO-d6) was used as the solvent for H NMR. The parameters of the UV spectrophotometer were as follows: the receiving wavelength was 350 nm to 700 nm. The parameters of the fluorescence spectrometer were as follows: the excitation wavelength was 423 nm, and the receiving wavelength was 600 nm to 750 nm.

[0056] Application Example 1

[0057] Fluorescence titration of copper ions of different concentrations in 100% aqueous solution using the copper ion fluorescent probe (MP1) prepared in Example 1: MP1 was dissolved in distilled water to prepare a solution with a concentration of 10 μM; copper nitrate was dissolved in distilled water to prepare aqueous solutions of different concentrations. After adding 1.0 mL of MP1 solution (10 μM) to a 10 mL volumetric flask, 1.0 mL of copper ion aqueous solution of different concentrations was added, and the solution was diluted to 10.0 mL with distilled water. After standing for 5 minutes, the fluorescence change spectrum of the solution with different concentrations of copper ions was measured by fluorescence spectroscopy. The results are shown in Figure 2. Figure 3 shown.

[0058] The parameters of the fluorescence spectrometer are as follows: the excitation wavelength is 423 nm; the receiving wave band is 575 nm to 775 nm.

[0059] Application Example 2

[0060] The copper ion fluorescent probe (MP1) prepared in Example 1 responds to the fluorescence enhancement signal of copper ions: Since copper ions can coordinate with nitrogen atoms in MP1, the photoinduced electron transfer (PET) effect in the original molecule is blocked, and a more rigid molecular plane is formed, the π electron conjugated system of the molecule is expanded, so that the probe shows a change in fluorescence signal enhancement. Therefore, an equal concentration of copper ion solution can be added to a certain concentration of MP1 molecular solution, and ultraviolet light is used for excitation, and the change in fluorescence intensity of the MP1 solution is observed with the naked eye. The specific process is as follows: MP1 is dissolved in distilled water to form a solution with a concentration of 10μM, and copper nitrate is dissolved in distilled water to form a solution of 10μM. 0.4mL (10μM) of the above-mentioned MP4 solution is added to a 5mL bacterial culture bottle, and then 0.4mL of a 10μM copper nitrate aqueous solution is added, and the volume is diluted to 4.0mL with distilled water. After standing for 5 minutes, the change in fluorescence intensity of the solution is observed with the naked eye by irradiation with a handheld ultraviolet lamp (excitation wavelength is 365nm). The results are as follows. Figure 4 As shown. Figure 4 It can be seen that under the irradiation of ultraviolet light, Cu 2+ This can cause the color of the MP1 solution to change from a non-fluorescent state to emit bright red fluorescence, indicating that the MP1 probe exhibits an enhanced fluorescence signal response to copper ions.

[0061] Application Example 3

[0062] Selective detection of copper ions in aqueous solution by the copper ion fluorescent probe (MP1) prepared in Example 1: Since only copper ions can selectively complex with nitrogen atoms, causing the porphyrin signal group to respond with a spectral signal, a certain concentration of MP1 probe molecule aqueous solution can be mixed with equal concentrations of different metal ion aqueous solutions and then reacted, and its selectivity for copper ions can be measured using fluorescence spectroscopy. The specific process is as follows: 0.4 mL (10 μM) of the MP1 aqueous solution prepared in Example 1 was added to a 5 mL culture bottle, and then 0.4 mL of an equivalent amount of aqueous solution of different metal ions was added respectively, and the volume was diluted to 4.0 mL with distilled water. After standing for 5 minutes, the fluorescence change spectrum after the probe molecule interacted with different metal ions was measured using fluorescence spectroscopy. The results are shown in the figure. Figure 5 As shown in Figure 2, only the addition of copper ions can cause the fluorescence intensity of the MP1 probe to increase at 647 nm, indicating that the MP1 probe has a high selectivity for copper ions ( Figure 5 ).

[0063] The parameters of the fluorescence spectrometer are as follows: the excitation wavelength is 423 nm; the receiving wave band is 575 nm to 775 nm.

[0064] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the concept of the present invention. These improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fluorescence-enhanced copper ion probe, characterized in that: The structural formula of the probe molecule is as follows: Where M = Zn 2+ .

2. A method for preparing the fluorescence-enhanced copper ion probe according to claim 1, characterized in that: The steps include: cis-MFTPPS and 2-hydrazinepyridine are dissolved in an organic solvent at a molar ratio suitable for the reaction, heated to reflux under nitrogen protection, and reacted for 8 to 12 hours. After cooling, the solvent is removed by distillation under reduced pressure. After separation by reverse phase chromatography, the solution is dialyzed in a 1000 kDa dialysis bag to obtain the fluorescence-enhanced copper ion probe. The structural formula of the cis-MFTPPS is as follows: Where M = Zn 2+ .

3. The method for preparing a fluorescence-enhanced copper ion probe according to claim 2, wherein: The molar ratio suitable for the reaction is cis-MFTPPS:2-hydrazinepyridine is 1:

3.

4. The method for preparing a fluorescence-enhanced copper ion probe according to claim 2, wherein: The organic solvent is N,N-dimethylformamide.

5. The method for preparing a fluorescence-enhanced copper ion probe according to claim 2, wherein: The concentration of cis-MFTPPS in the solution was 0.025 mmol / mL.

6. The method for preparing a fluorescence-enhanced copper ion probe according to claim 2, wherein: The eluent for reverse phase chromatography separation is a methanol-water system, and the elution gradient ranges from 0% to 85% by volume of methanol.

7. Use of the fluorescence-enhanced copper ion probe according to claim 1 for fluorescence detection of trace copper ions in 100% aqueous solution.

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