Application of Multifunctional Ratiometric Fluorescent Probe in the Detection of pH, Copper Ions and Sulfide Ions

Abstract

The present invention discloses a multifunctional ratio fluorescence probe, which is mainly obtained by the coupling reaction of a metal-organic framework material Uio-66-NH2 and protoporphyrin. The preparation method may include the following steps: Step 1, synthesis of the metal-organic framework material Uio-66-NH2; Step 2, preparation of a protoporphyrin stock solution; Step 3, coupling action of Uio-66-NH2 and protoporphyrin. The multifunctional fluorescence probe of the present invention is simple to prepare, has a very significant fluorescence response to acidity and alkalinity, has an obvious visual effect under the irradiation of a 365 nm ultraviolet lamp, and is easy to distinguish. In addition, the probe can realize highly sensitive reversible detection of copper ions and sulfide ions, and has the advantages of high selectivity, rapid detection, strong practicability, etc., providing a potential application prospect for the rapid detection of target substances in the water environment.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence detection, and particularly relates to a multifunctional ratio fluorescence probe and its application in the detection of pH value, copper ions and sulfide ions. Background Art

[0002] The change of pH value in water directly affects the chemical reactions, biological activities and toxicity of pollutants in water. For example, in an alkaline environment, the degradation rate of organic matter will slow down, causing the problem of water eutrophication; fish and plankton are very sensitive to the change of pH value, and too high or too low pH value will cause fish asphyxiation, abnormal development of fish eggs and death of plankton, thus destroying the balance of the ecosystem; heavy metal ions are more likely to be released in an acidic environment, increasing the exposure risk of organisms, while some pesticides will degrade more slowly in an alkaline environment, increasing the pollution risk to water. Therefore, accurately monitoring and controlling the pH value of the water environment is of great significance for maintaining the health of aquatic organisms and ecological balance.

[0003] Copper ions are one of the commonly existing metal ions. Excessive copper ions are released into the environment in various ways, such as the production of electrical equipment, batteries, industrial machinery, etc. Copper ions have toxic effects on microorganisms and aquatic plants in water. High concentrations of copper ions will inhibit the growth and metabolic activities of microorganisms in water, reducing biodiversity and the stability of the ecosystem. In addition, copper ions will also have an adverse impact on the growth and photosynthesis of aquatic plants, destroying the plant community structure in water and affecting the degree of water eutrophication. Research shows that excessive intake of copper ions may cause a series of damages to human health.

[0004] Sulfide ions widely exist in wastewater and surface water. This traditional pollutant not only comes from industrial production, but also can be produced in the biological metabolic process. Excessive accumulation of sulfide will cause various serious harms to the ecological environment and organisms. Protonated HS - and H2S are more toxic and corrosive. As the third physiological gas transmitter in addition to CO and NO, H2S participates in many physiological and pathological processes. The level of hydrogen sulfide in the blood of healthy people is 10 - 100 μM. Currently, it is found that diseases such as Alzheimer's disease, diabetes, Down syndrome and cancer are closely related to the activities of hydrogen sulfide in the human body.

[0005] The detection of pH value can be determined by a pH meter, while traditional copper ion sensing technology consists of various precision instruments, such as ICP-AES, ICP-MS, and electrochemical analysis. Conventional detection methods for sulfide ions include titration, spectrophotometry, electrochemistry, and gas chromatography. These traditional methods usually require expensive and precise instruments, complex sample pretreatment, and are very time-consuming. In contrast, fluorescence method has been proven to be a more powerful technique for detecting low-concentration targets due to its unique advantages of high sensitivity, specificity, and low cost. In particular, the development of ratio fluorescence sensors has attracted increasing attention. Different from traditional single-intensity-based fluorescence sensors, ratio fluorescence eliminates the interference of irrelevant factors through the changes in multiple emission peaks, providing color discrimination that can be recognized by the naked eye while improving sensitivity. Therefore, it is of great significance to develop a ratio fluorescence strategy to achieve efficient and intuitive in-situ detection of the above-mentioned target substances. Summary of the Invention

[0006] The purpose of the present invention is to provide a multifunctional ratio fluorescence probe and its application in the detection of acidity, copper ions, and sulfide ions.

[0007] The multifunctional ratio fluorescence probe described in the present invention is mainly obtained by the coupling reaction of metal-organic framework material Uio-66-NH2 and protoporphyrin. Its preparation method may include the following steps: Step 1, synthesis of metal-organic framework material Uio-66-NH2; Step 2, preparation of protoporphyrin stock solution; Step 3, coupling effect of Uio-66-NH2 and protoporphyrin.

[0008] In the described preparation method, for the metal-organic framework material Uio-66-NH2 in Step 1, it can be a commercially available product or obtained through the following preparation method: 0.234 g of zirconium chloride is added to 35 mL of N,N-dimethylformamide, and then 0.182 g of 2-aminoterephthalic acid is added. The mixed solution is ultrasonically dissolved for 5 min and then transferred to a 60 mL Teflon-lined stainless steel autoclave for solvothermal reaction in an oven (120 °C, 1440 min). After cooling to room temperature, the brown-yellow crystals are collected by centrifugation (10000 rpm, 8 min) and washed several times with N,N-dimethylformamide and ethanol to remove unreacted substances. Finally, it is placed in a vacuum drying oven (60 °C) overnight, and the obtained brown-yellow powder is weighed after grinding. The 100 μg / mL Uio-66-NH2 stock solution is stored at 4 °C in the dark for later use, and the solvent is 2 mM, pH = 7 phosphate buffer solution.

[0009] Preparation method of multifunctional ratio fluorescence probe of the present invention: Equal volumes of Uio-66-NH2 stock solution and protoporphyrin solution are mixed and treated by ultrasonic treatment to obtain a probe solution; the concentration of the Uio-66-NH2 stock solution is 50-150 μg / mL, preferably 80-120 μg / mL, and the solvent is 2 mM phosphate buffer solution with pH = 7; the protoporphyrin solution is an N,N-dimethylformamide solution of protoporphyrin with a concentration of 100-300 μM, preferably 150-250 μM.

[0010] In the described preparation method, preferably, a Uio-66-NH2 stock solution with a concentration of 100 μg / mL and a protoporphyrin solution with a concentration of 200 μM are mixed in equal volumes and treated by ultrasonic treatment for 5-15 min.

[0011] The multifunctional ratio fluorescence probe obtained by the described preparation method can be applied in pH detection. Specifically, the following method steps can be adopted:

[0012] (1) Prepare an aqueous solution with a pH value of 2-12 using hydrochloric acid and sodium hydroxide. Take 1900 μL of the aqueous solution with different pH values into a centrifuge tube, and then add 100 μL of the probe solution, shake well, and perform fluorescence detection after 5-8 min. The excitation wavelength is 365 nm, the scanning speed is 1000-1300 nm / min, and the slit width is 5 nm for both. Record the emission spectrum at 400-700 nm.

[0013] (2) Establish a relationship curve between the fluorescence emission intensity ratio F 629 / F 431 and pH.

[0014] (3) Take 1900 μL of the solution to be tested and perform the operation process described in step (1).

[0015] (4) According to the relationship curve between the fluorescence emission intensity ratio F 629 / F 431 and pH in step (2), obtain the pH value of the solution to be tested.

[0016] As another preferred scheme, the application can also adopt the following method steps:

[0017] (1) Prepare an aqueous solution with a pH value of 2-12 using hydrochloric acid and sodium hydroxide. Take 1900 μL of the aqueous solution with different pH values into a four-sided light-transmitting cuvette, and then add 100 μL of the probe solution, shake well, and take an image with a smartphone after 5-8 min. Use software to extract the RGB values of the picture.

[0018] (2) Establish a relationship curve between the ratio R / B value of the red channel value to the blue channel value and the pH value.

[0019] (3) Replace the aqueous solution with different pH values with 1900 μL of the test solution to perform the operation described in step (1).

[0020] (4) Obtain the pH value of the test solution according to the relationship curve between the R / B value and pH in step (2).

[0021] The multifunctional ratio fluorescence probe obtained by the preparation method of the present invention can also be used in the detection of copper ions. Specifically, the following method steps can be adopted:

[0022] (1) In a 2 mL system, the volume of the probe solution is 100 μL. Add 1900 μL of copper 2+ solutions with different concentrations, shake well, and perform fluorescence detection after 5 - 8 min. The excitation wavelength is 365 nm, the scanning speed is 1200 nm / min, and the slit width is 5 nm for both. Record the emission spectrum from 400 - 700 nm.

[0023] (2) Establish a curve of the fluorescence ratio F 629 / F 431 versus the concentration of copper 2+ ;

[0024] (3) Take 1900 μL of the test solution containing copper 2+ to replace the copper 2+ solutions with different concentrations and perform the operation described in step (1).

[0025] (4) According to the curve of the fluorescence ratio F 629 / F 431 versus the concentration of copper 2+ , obtain the concentration value of copper 2+ in the test solution.

[0026] The multifunctional ratio fluorescence probe obtained by the preparation method of the present invention can also be used in the detection of sulfide ions. Specifically, the following method steps can be adopted:

[0027] (1) In a 2 mL system, the volume of the probe solution is 100 μL. First, add 1850 μL of a 5 μM copper 2+ solution and incubate for 5 - 8 min. Then add 50 μL of sulfide 2- solutions with different concentrations, shake well, and perform fluorescence detection after 5 - 8 min. The excitation wavelength is 365 nm, the scanning speed is 1200 nm / min, and the slit width is 5 nm for both. Record the emission spectrum from 400 - 700 nm.

[0028] (2) Establish a curve of the fluorescence ratio F 629 / F 431 versus the concentration of sulfide 2- ;

[0029] (3) Take 50 μL of the test solution containing S 2- to replace the S solutions with different concentrations and perform the operation process described in step (1); 2-

[0030] (4) According to the fluorescence ratio F 629 / F 431 and the curve of the S concentration, obtain the S concentration value of the test solution. 2- 2-

[0031] Based on the different responses of Uio-66-NH2 and protoporphyrin to different pH values, that is, Uio-66-NH2 emits blue or cyan fluorescence under acidic conditions, while protoporphyrin emits red fluorescence under alkaline conditions. Therefore, the ratio fluorescence mechanism formed by the two can be used for quantitative detection and semi-quantitative visual analysis of pH values. Paramagnetic copper ions are easy to form relatively stable metal complexes with azacyclic compounds, and significantly quench the fluorophore through the mechanism of electron transfer or energy transfer. The formation of copper-protoporphyrin complexes can quench the red fluorescence of protoporphyrin, while the blue fluorescence of Uio-66-NH2 is not affected. Therefore, this ratio system can be used for quantitative analysis of copper ions. In the presence of copper ions, based on the solubility product constant of copper sulfide being 1.27×10 -36 , sulfide ions have the ability to displace copper ions from copper-protoporphyrin complexes and turn on the red fluorescence of protoporphyrin again. By measuring the fluorescence intensity ratio F 629 / F 431 at 431 nm and 629 nm, quantitative analysis of copper ions and sulfide ions can be carried out.

[0032] Based on the coupling of nano metal-organic framework (Uio-66-NH2) and protoporphyrin, the present invention prepares a ratio fluorescence detection probe. The probe has obvious color changes at different pH values, and the color signal R / B value has an excellent linear relationship with pH (3 - 10). The response time of the probe to copper ions and sulfide ions is extremely fast, and obvious color changes can be observed within 30 s. The detection limit of copper ions is 55 nM, and the detection limit of sulfide ions is 35 nM. In addition, the probe exhibits excellent selectivity, reversibility, and practicability.

[0033] The multifunctional fluorescence probe of the present invention is simply prepared, has a very significant fluorescence response to acidity and alkalinity, has an obvious visual effect under ultraviolet light at 365 nm, and is easy to distinguish. In addition, the probe can achieve highly sensitive reversible detection of copper ions and sulfide ions, and has the advantages of high selectivity, rapid detection, and strong practicability, providing potential application prospects for the rapid detection of target substances in water environment. Description of the Drawings

[0034] Figure 1 ​​​is the fluorescence spectrum of the probe Uio-66-NH2 / Ppix at different pH values (2 - 12);

[0035] Figure 2 In it, the ratio of the red channel value to the blue channel value (R / B) has a linear relationship with the pH value (3 - 10);

[0036] Figure 3 is the probe Uio-66-NH2 / Ppix for Cu 2+ and Uio-66-NH2 / Ppix / Cu 2+ for S 2- response time curve;

[0037] Figure 4 is the fluorescence spectrum of Uio-66-NH2 / Ppix at different Cu 2+ concentrations (0.1 - 10 μM);

[0038] Figure 5 is the fluorescence intensity ratio F 629 / F 431 versus Cu 2+ concentration correlation curve;

[0039] Figure 6 is the fluorescence spectrum of the probe solution Uio-66-NH2 / Ppix / Cu 2- at different S 2+ concentrations (0 - 90 μM);

[0040] Figure 7 is the influence of different S 2- concentrations (10 - 500 μM) on F 629 / F 431 ;

[0041] Figure 8 is the probe selectivity experiment and anti-interference experiment for S 2- ;

[0042] Figure 9 is the schematic diagram of the fluorescence principle of the probe of the present invention; Specific Embodiments

[0043] The following embodiments are further descriptions of the content of the present invention to illustrate the technical content of the present invention. However, the substantial content of the present invention is not limited to what is described in the following embodiments. Those of ordinary skill in the art can and should know that any simple changes or substitutions based on the substantial spirit of the present invention should fall within the protection scope required by the present invention.

[0044] Example 1

[0045] A probe solution was obtained by mixing an equal volume of a 100 μg / mL Uio-66-NH2 stock solution with a 200 μM protoporphyrin (Ppix) solution in a brown color and subjecting the mixture to ultrasonic treatment for 10 min. The solvent used for the Uio-66-NH2 stock solution was a 2 mM phosphate buffer solution with a pH of 7; the protoporphyrin solution was an N,N-dimethylformamide solution of protoporphyrin.

[0046] Fluorescent response of the fluorescent probe Uio-66-NH2 / Ppix to different pH values:

[0047] Aqueous solutions with pH values ranging from 2 to 12 were prepared using hydrochloric acid and sodium hydroxide. 1900 μL of the aqueous solutions with different pH values were taken into centrifuge tubes, and then 100 μL of the probe solution was added. After shaking well, fluorescence detection was performed after 5 min. The excitation wavelength was 365 nm, the scanning speed was 1200 nm / min, and the slit width was 5 nm for both. The emission spectra in the range of 400 - 700 nm were recorded as Figure 1 shown. The direction of the arrow in the figure is the direction of increasing pH.

[0048] It was observed that when the pH value was less than 3, only the fluorescence of Uio-66-NH2 (460 nm) was shown, and the solution appeared cyan; when pH = 3, the emission peak blue-shifted to 440 nm, and the solution appeared blue; when the pH value was 4 - 12, the emission peak of Uio-66-NH2 was basically at 431 nm, while protoporphyrin began to emit red fluorescence (629 nm). Further, it was found that F 629 / F 431 followed the equation at pH values of 4 - 10: F 629 / F 431 = 0.046 * exp(0.442 * [pH]) - 0.254 (R 2 = 0.992). Therefore, this probe can be used for the quantitative detection of pH values.

[0049] Example 2

[0050] Response of the color signal R / B to different pH values:

[0051] Aqueous solutions with pH values ranging from 2 to 12 were prepared using hydrochloric acid and sodium hydroxide. 1900 μL of the aqueous solutions with different pH values were taken into a four-sided light-transmitting cuvette, and then 100 μL of the probe solution was added. After shaking well, an image was taken using a smartphone after 5 min, and the RGB values of the picture were extracted using software. As Figure 2 shown, the ratio (R / B) of the red channel value to the blue channel value had an excellent linear relationship with pH (3 - 10) (R 2 = 0.995). Therefore, this probe can be used for visual detection of pH values.

[0052] Example 3

[0053] Kinetic tests of the probe Uio-66-NH2 / Ppix for Cu 2+ and Uio-66-NH2 / Ppix / Cu 2+ for S 2- :

[0054] Prepare 1 mM Cu 2+ and 10 mM S 2- solutions for standby. In the fluorescence detection system, the concentrations of Cu 2+ and S 2- are 5 μM and 10 μM respectively, the added volumes are 10 μL and 20 μL respectively, the probe solution is 100 μL, and it is in 1900 μL of ultrapure water. The parameters of the fluorescence spectrometer are excitation wavelength 365 nm, scanning speed 1200 nm / min, and slit width 5 nm for both, and the emission spectrum in the range of 400 - 700 nm is recorded. Immediately after adding Cu 2+ , detect the fluorescence intensities at 431 nm and 629 nm, and then detect once every 30 s. After 5 min, add S 2- , and do the same.

[0055] Establish a curve of the fluorescence ratio F 629 / F 431 versus time. As Figure 3 shown, within about 30 s, Cu 2+ almost quenched the fluorescence at 629 nm; while after adding S 2- , the fluorescence at 629 nm was immediately restored and enhanced within 30 s, and the kinetic performance is very excellent. Therefore, using this probe to detect Cu 2+ and S 2- is very fast.

[0056] Example 4

[0057] Titration detection of the fluorescence probe Uio-66-NH2 / Ppix for different concentrations of Cu 2+ :

[0058] In a 2 mL ultrapure water system, the volume of the probe solution is 100 μL, and 1900 μL of Cu 2+ with different concentrations (0, 0.1, 0.2, 0.5, 0.8, 1, 2, 3, 5, 8, 10 μM) is added. Shake well and perform fluorescence detection after 5 min. The excitation wavelength is 365 nm, the scanning speed is 1200 nm / min, and the slit width is 5 nm for both. Record the emission spectrum in the range of 400 - 700 nm. As Figure 4 shown, as the concentration of Cu 2+ increases (i.e., in the direction of the arrow in the figure), the fluorescence of Uio-66-NH2 increases slightly and then remains relatively constant, the fluorescence of protoporphyrin gradually weakens, and at the same time the fluorescence intensity ratio F629 / F 431 also decreases, and the probe solution changes from red to blue.

[0059] Establish the fluorescence ratio F 629 / F 431 versus the concentration of Cu 2+ , as Figure 5 shown, the fluorescence of protoporphyrin can be almost completely quenched by one equivalent of Cu 2+ (5 μM). When the concentration of Cu 2+ is greater than 5 μM, F 629 / F 431 remains constant, indicating that Cu 2+ and protoporphyrin bind in a 1:1 molar ratio. By plotting the relationship between F 629 / F 431 and the concentration of Cu 2+ (0.1 - 10 μM) and performing non-linear fitting, the equation F 629 / F 431 = 0.916 * exp(-0.82 * [Cu 2+ ) + 0.015 (R 2 = 0.994) is obtained. Further, it is found that there is a good linear relationship between F 629 / F 431 and the concentration of Cu 2+ in the range of 0.1 - 2 μM, and the fitting equation is: F 629 / F 431 = -0.302 * [Cu 2+ + 0.847 (R 2 = 0.997). According to the formula LOD = 3σ / S, where σ is the overall standard deviation of the F 629 / F 431 values of 11 prepared probe solutions, which is 0.005507, and S is the slope of the fitting equation, which is 0.302, the detection limit of Cu 2+ is calculated to be 55 nM. Therefore, this probe can be used for the quantitative detection of Cu 2+ .

[0060] Example 5

[0061] Titration detection of the fluorescence probe Uio-66-NH2 / Ppix / Cu 2+ against different concentrations of S 2- :

[0062] In a 2 mL ultrapure water system, the volume of the probe solution is 100 μL. First, add 1850 μL of Cu 2+ with a concentration of 5 μM and incubate for 5 min, then add 50 μL of S 2- with different concentrations.Make their concentrations 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μM, shake well, and perform fluorescence detection after 5 min. The excitation wavelength is 365 nm, the scanning speed is 1200 nm / min, and the slit width is 5 nm for all. Record the emission spectrum from 400 - 700 nm. As Figure 6 shown, in the presence of S 2- , the blue fluorescence intensity at 431 nm decreases by about 27%, and the fluorescence at 629 nm continuously increases as the concentration of S 2- increases (i.e., in the direction of the arrow in the figure), and at this time the solution changes from blue to red.

[0063] Establish a curve of the fluorescence ratio F 629 / F 431 versus the concentration of S 2- . As Figure 7 shown, when the concentration of S 2- is greater than 90 μM, the F 629 / F 431 value basically remains unchanged. The alkalinity of S 2- and the formation of CuS lead to the enhancement of the fluorescence at 629 nm, causing the protoporphyrin quenched by Cu 2+ to re - emit red fluorescence. The F 629 / F 431 value has an excellent linear correlation with the concentration of S 2- in the range of 10 - 90 μM, with its R 2 = 0.998, and the fitting equation is F 629 / F 431 = 0.028 * [S 2- - 0.252. The detection limit LOD = 35 nM is calculated. Therefore, this probe can be used for the quantitative detection of S 2- .

[0064] Example 6

[0065] Selectivity and interference of the fluorescent probe Uio - 66 - NH2 / Ppix / Cu 2+ towards different amino acids and anions:

[0066] Prepare common anions (WO3 2- , IO3 - , HPO4 2- , H2PO4 - , Cl -1 , Br -1 , I -1 , HCO3 - , Ac - , NO2 - , SO4 2- , HSO3 -,S2O3 2- ,S 2- ) and amino acids (Cys, Phe, Ala, Arg, Trp, Gly, Thr, Pro, Ser) for later use. In 2 mL ultrapure water system, add 100 μL probe solution and 1850 μL 5 μM Cu 2+ Incubate for 5 min, then add 50 μL of 10 mM S 2- The other 22 anions and amino acids were mixed and shaken. After 5 minutes, fluorescence detection was performed with an excitation wavelength of 365 nm, a scan rate of 1200 nm / min, a slit width of 5 nm, and the emission spectrum from 400 to 700 nm was recorded. Subsequently, 20 μL of 10 mM S 2- , shake well, and perform fluorescence detection after 5 minutes.

[0067] like Figure 8 As shown, only when S 2- When sulfhydryl compound cysteine was present, the fluorescence emission peak at 629 nm changed significantly, with the intensity increasing by about 100 times the initial value, while it was only 5 times after treatment with cysteine, indicating that the probe can distinguish inorganic sulfide from organic thiols. 2- When coexisting, F 629 / F 431 Value and S 2- F when it exists alone 629 / F 431 The values are comparable, even if the concentration of other interfering substances is S 2- These results show that Uio-66-NH2 / Ppix / Cu 2+ For S 2- It has good selectivity and strong anti-interference ability.

[0068] It should be noted that the above-mentioned technical contents of the present invention are only for explanation and clarification to enable those skilled in the art to understand the technical essence of the present invention, so the technical contents are not used to limit the substantial protection scope of the present invention. The substantial protection scope of the present invention shall be subject to the claims. Those skilled in the art should know that any modification, equivalent substitution and improvement based on the substantial spirit of the present invention shall be within the substantial protection scope of the present invention.

Claims

1. A preparation method of a multifunctional ratio fluorescence probe, characterized in that, Mix the Uio-66-NH2 stock solution and the protoporphyrin solution in equal volumes and treat them by ultrasonic treatment to obtain a probe solution; the concentration of the Uio-66-NH2 stock solution is 50-150 μg / mL, and the solvent is a phosphate buffer solution with a concentration of 2 mM and a pH of 7. The protoporphyrin solution is an N,N-dimethylformamide solution of protoporphyrin with a concentration of 100-300 μM.

2. The preparation method according to claim 1, characterized in that, The concentration of the Uio-66-NH2 stock solution is 80-120 μg / mL.

3. The preparation method according to claim 1, characterized in that, The concentration of the protoporphyrin solution is 150-250 μM.

4. The preparation method according to claim 1, characterized in that, Mix the Uio-66-NH2 stock solution with a concentration of 100 μg / mL and the protoporphyrin solution with a concentration of 200 μM in equal volumes and perform ultrasonic treatment for 5-15 min.

5. A multifunctional ratio fluorescence probe obtained by the preparation method according to any one of claims 1-4.

6. The application of the multifunctional ratio fluorescence probe according to claim 5 in pH detection.

7. The application according to claim 6, wherein Adopt the following method steps: (1) Prepare an aqueous solution with a pH value of 2-12 using hydrochloric acid and sodium hydroxide. Take 1900 μL of the aqueous solution with different pH values into a centrifuge tube, and then add 100 μL of the probe solution, shake well, and perform fluorescence detection after 5-8 min. The excitation wavelength is 365 nm, the scanning speed is 1000-1300 nm / min, and the slit width is 5 nm. Record the emission spectrum at 400-700 nm. (2) Establish the relationship curve between the fluorescence emission intensity ratio F 629 / F 431 and pH; (3) Take 1900 μL of the test solution and perform the operation process described in step (1). (4) Based on the relationship curve of the fluorescence emission intensity ratio F 629 / F 431 and pH, the pH value of the solution to be measured is obtained.

8. The application according to claim 6, characterized in that Adopt the following method steps: (1) Prepare an aqueous solution with a pH value of 2-12 using hydrochloric acid and sodium hydroxide. Take 1900 μL of the aqueous solution with different pH values into a four-side light-transmitting cuvette, and then add 100 μL of the probe solution, shake well, and take an image after 5-8 min. Use software to extract the RGB values of the picture. (2) Establish a relationship curve between the ratio R / B value of the red channel value and the blue channel value and the pH value. (3) Take 1900 μL of the test solution and perform the operation process described in step (1). (4) According to the relationship curve between the R / B value and the pH value in step (2), obtain the pH value of the test solution.

9. The application of the multifunctional ratio fluorescence probe according to claim 5 in copper ion detection.

10. The application according to claim 9, wherein, Adopt the following method steps: In a 2 mL system, the volume of the probe solution is 100 μL, and 1900 μL of Cu solutions with different concentrations are added. After shaking well, fluorescence detection is carried out after 5 - 8 min. The excitation wavelength is 365 nm, the scanning speed is 1200 nm / min, and the slit width is 5 nm for both. The emission spectrum in the range of 400 - 700 nm is recorded. 2+ In a 2 mL system, the volume of the probe solution is 100 μL, and 1900 μL of Cu solutions with different concentrations are added. After shaking well, fluorescence detection is carried out after 5 - 8 min. The excitation wavelength is 365 nm, the scanning speed is 1200 nm / min, and the slit width is 5 nm for both. The emission spectrum in the range of 400 - 700 nm is recorded. (2)Establish the fluorescence ratio F 629 / F 431 versus the curve of Cu 2+ concentration; (3) Take 1900 μL of the test solution and perform the operation process described in step (1). (4) According to the fluorescence ratio F 629 / F 431 and the curve of Cu 2+ concentration, obtain the Cu 2+ concentration value of the solution to be measured.

11. The application of the multifunctional ratio fluorescence probe according to claim 5 in sulfide ion detection.

12. The application according to claim 11, wherein Adopt the following method steps: (1) In a 2 mL system, the volume of the probe solution is 100 μL. First, add 1850 μL of a 5 μM Cu 2+ solution and incubate for 5 - 8 minutes. Then, add 50 μL of S 2- solutions with different concentrations, shake well, and perform fluorescence detection after 5 - 8 minutes. The excitation wavelength is 365 nm, the scanning speed is 1200 nm / min, and the slit width is 5 nm for both. Record the emission spectrum from 400 - 700 nm; (2) Establish the fluorescence ratio F 629 / F 431 versus the curve of the concentration of S 2- ; (3)Take 50 μL of the test solution containing S 2- and perform the operation procedure described in step (1); (4) According to the fluorescence ratio F 629 / F 431 versus the curve of the S 2- concentration, the S 2- concentration value of the solution to be measured is obtained.

Images