Fluorescent molecular probe for detecting copper ions as well as preparation method and application of fluorescent molecular probe

Abstract

The invention provides a fluorescent molecular probe for copper ion detection and a preparation method and application thereof, and belongs to the technical field of heavy metal detection.The preparation method of the fluorescent molecular probe for copper ion detection comprises the following steps that salicylaldehyde and N, N '-bis (2-aminoethyl)-2, 6-pyridinedicarboxamide are subjected to a condensation reaction, and picolinamide bis-Schiff base is obtained; and reducing the picolinamide bis-Schiff base by using a reducing agent to obtain the fluorescent molecular probe for copper ion detection. High-selectivity recognition of Cu < 2 + > in a water environment system can be achieved, interference of other metal ions in an aqueous solution is avoided, and the high anti-interference capacity is achieved. The method can be applied to detection of Cu < 2 + > pollution in a water system, and trace Cu < 2 + > in a water phase can be sensitively detected.

Description

Technical Field

[0001] The present application relates to the technical field of heavy metal detection, and in particular to a fluorescent molecular probe for copper ion detection, and a preparation method and use thereof. Background Art

[0002] Copper is an indispensable micronutrient for human health. It activates the formation of hemoglobin and promotes the absorption and utilization of iron. It is of great significance in the transfer of electrons, the synthesis of elastin, the metabolism of connective tissue, purine metabolism, the formation of phospholipids and nerve tissue. However, excessive intake or accumulation of copper can also cause certain harm to the body, such as protein denaturation, liver cirrhosis, diarrhea, vomiting, movement disorders and sensory nerve disorders. With the rapid development of the industrial market, copper has been widely used in all walks of life. Copper has become increasingly polluting the environment and causing increasing harm to human health. Therefore, copper is an important detection indicator in water, soil, food, and organisms. Its concentration directly affects the growth and development of individual organisms and environmental safety. How to safely and quickly determine trace copper elements in the environment has positive scientific significance.

[0003] The researchers designed a Cu 2+ Most fluorescent molecular probes are of the fluorescence quenching type, but the fluorescence quenching type probes have the disadvantages of large detection background signal, low sensitivity, and are not suitable for biological sample detection. In addition, the existing fluorescent molecular probes also have the problems of poor water solubility and poor selectivity. Summary of the invention

[0004] In view of the technical problems existing in the background technology, the present application provides a fluorescent molecular probe for copper ion detection, and its preparation method and use, aiming to solve the technical problems that fluorescent quenching probes have large detection background signals, low sensitivity, and are not suitable for biological sample detection, and existing fluorescent molecular probes also have poor water solubility and poor selectivity.

[0005] In a first aspect, the embodiments of the present application provide a fluorescent molecular probe for copper ion detection, and the structural formula of the fluorescent molecular probe for copper ion detection is: .

[0006] In a second aspect, the present invention provides a method for preparing a fluorescent molecular probe for copper ion detection, comprising the following steps: (1) Salicylaldehyde reacts with N,N'-bis(2-aminoethyl)-2,6-pyridinedicarboxamide to obtain pyridineamide bis-Schiff base; (2) Using a reducing agent to reduce the pyridinamide bis-Schiff base to obtain a pyridinamide bis-Schiff base secondary amine fluorescent molecular probe.

[0007] In some embodiments, in step S1, salicylaldehyde and N,N'-bis(2-aminoethyl)-2,6-pyridinedicarboxamide are added to an alcohol solvent, heated to reflux at 65-80°C, reacted for 5-7h, and then the heating is stopped and cooled to room temperature to obtain a pyridineamide bis-Schiff base alcohol solution.

[0008] In some embodiments, the alcohol solvent includes one or more of methanol, ethanol, and isopropanol.

[0009] In some embodiments, the reducing agent is NaBH 4 , NaBH 3 CN and Na(OAc) 3 One or more of BH; the molar ratio of the reducing agent to the pyridineamide bis-Schiff base is (3~5):1.

[0010] In some embodiments, in step S2, the pyridineamide bis-Schiff base alcohol solution is cooled to below 20° C. using an ice-water bath or an ice-salt bath, the reducing agent is dispersed in methanol or ethanol to obtain a reducing agent solution, the reducing agent solution is added to the cooled pyridineamide bis-Schiff base alcohol solution, the reaction is stirred for more than 12 hours, the solvent is evaporated, water is added to stir the reaction, and then the crude product of pyridineamide bis-Schiff base secondary amine is obtained after extraction, washing, and drying to remove the solvent.

[0011] In some embodiments, the crude product of pyridineamide bis-Schiff base secondary amine is recrystallized using ethanol, and the precipitated crystals are filtered to obtain a fluorescent molecular probe for copper ion detection.

[0012] In a third aspect, the present invention provides a method for detecting copper ions using a fluorescent molecular probe. 2+ Conduct qualitative or quantitative analysis.

[0013] In some embodiments, a fluorescent molecular probe for copper ion detection is used to detect Cu 2+ The qualitative analysis includes the following steps: Dispersing the fluorescent molecular probe in deionized water to obtain a fluorescent molecular probe solution, wherein the concentration of the fluorescent molecular probe is 7.5-10 μmol / L; The fluorescent molecular probe and the sample to be tested are mixed evenly, the metal ion concentration in the sample to be tested is more than twice the molar concentration of the fluorescent probe, and the mixture is reacted for more than 5 minutes to obtain a sample solution; The fluorescence intensity A1 of the fluorescent molecular probe solution is detected by a fluorescence spectrometer at an emission wavelength of 310 nm, and then the fluorescence intensity A2 of the sample solution is detected. If A2 is greater than or equal to 7A1, it can be determined that the sample contains Cu. 2+Otherwise, it is determined that the sample does not contain Cu. 2+ .

[0014] In some embodiments, the fluorescent molecular probe is used to detect Cu 2+ The quantitative analysis includes the following steps: Prepare the gradient concentration of Cu 2+ Solution, Cu 2+ Fluorescent probes were added to the solution to detect the 2+ The fluorescence intensity of the solution at an emission wavelength of 310 nm was used to construct a relationship between the fluorescence intensity and Cu 2+ Standard curve of concentration; The fluorescent molecular probe and the sample to be tested are mixed evenly and reacted for more than 5 minutes to obtain a sample solution; The fluorescence intensity of the sample solution at an emission wavelength of 310 nm was detected. 2+ The concentration of Cu in the sample solution was calculated using the standard curve of concentration changes. 2+ Content; Cu 2+ The detection limit was 2.69×10 -8 mol / L.

[0015] Different from the existing technical solutions, the beneficial effects of this application include: The present application provides a pyridineamide bis-Schiff base secondary amine fluorescent molecular probe for copper ion detection, which is a Cu containing pyridine fluorescent group. 2+ Fluorescent molecular probe, which can detect Cu in water environment system 2+ It has high selective recognition, is not interfered by other metal ions in aqueous solution, and has strong anti-interference ability. 2+ In the detection of pollution, it can sensitively detect trace amounts of Cu in the water phase 2+ (Detection limit is 2.69×10 -8 mol / L, complex constant 1.28×10 8 (mol / L) -1 ), and has low toxicity, so it has great application value in the fields of chemical industry, environment, biomedicine, etc. and has been initially applied to trace Cu in the environment 2+ The fluorescent molecular probe can detect Cu 2+ Afterwards, the fluorescence enhancement effect can be shown in the pH range of 5 to 9. The fluorescent molecular probe recognizes the response very quickly and can complete the detection of Cu in the water system environment within 5 minutes. 2+ The synthetic raw materials of the fluorescent molecular probe are simple and easy to obtain, the cost is low, the conditions are mild, the steps are simple, the separation and purification of the product are simple, and the yield is as high as 69-71%.

[0016] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings used in the present application. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 is the fluorescence emission spectrum of the fluorescent molecular probe L in aqueous solution; Figure 2 The selective fluorescence emission spectra of the fluorescent molecular probe L and each metal ion; Figure 3 The fluorescent molecular probe L and different concentrations of Cu 2+ Fluorescence emission spectrum of Figure 4 The fluorescent molecular probe L was used to determine Cu 2+ The standard curve of Figure 5 is the Benesi-Hildebrand linear analysis curve and complex constant of the fluorescence emission of the fluorescent molecular probe L; Figure 6 For the fluorescent molecular probe L and Cu 2+ Fluorescence emission graphs in solutions with different pH values; Figure 7 For the fluorescent molecular probe L and Cu 2+ Fluorescence emission graphs in response at different times; Figure 8 Cu 2+ Fluorescence emission diagrams under the interference of different metal ions. DETAILED DESCRIPTION

[0019] The following embodiments of the technical solution of the present application are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0021] In the description of the embodiments of the present application, the technical terms "first", "second", etc. are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features. In the description of the embodiments of the present application, the meaning of "multiple" is more than two, unless otherwise clearly and specifically defined.

[0022] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0023] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0024] In the description of the embodiments of the present application, the term "multiple" refers to more than two (including two). Similarly, "multiple groups" refers to more than two groups (including two groups), and "multiple pieces" refers to more than two pieces (including two pieces).

[0025] The researchers designed a Cu 2+ Most fluorescent molecular probes are of the fluorescence quenching type, but the fluorescence quenching type probes have the disadvantages of large detection background signal, low sensitivity, and are not suitable for biological sample detection. In addition, the existing fluorescent molecular probes also have the problems of poor water solubility and poor selectivity.

[0026] In order to solve the problems that fluorescence quenching probes have large detection background signals, low sensitivity, and are not suitable for biological sample detection, and that existing fluorescent molecular probes have poor water solubility and poor selectivity, the present application provides a fluorescent molecular probe for copper ion detection and a preparation method and use thereof, which can realize the detection of Cu ions in a water environment system. 2+It has high selective recognition, is not interfered by other metal ions in aqueous solution, and has strong interference ability.

[0027] In a first aspect, the embodiments of the present application provide a fluorescent molecular probe for copper ion detection, and the structural formula of the fluorescent molecular probe for copper ion detection is: .

[0028] In a second aspect, the present invention provides a method for preparing a fluorescent molecular probe for copper ion detection, comprising the following steps: (1) Salicylaldehyde reacts with N,N'-bis(2-aminoethyl)-2,6-pyridinedicarboxamide to obtain pyridineamide bis-Schiff base; (2) Using a reducing agent to reduce the pyridinamide bis-Schiff base, a pyridinamide bis-Schiff base secondary amine fluorescent molecular probe (L) is obtained.

[0029] In some embodiments, in step S1, salicylaldehyde and N,N'-bis(2-aminoethyl)-2,6-pyridinedicarboxamide are added to an alcohol solvent, heated to reflux at 65-80°C, reacted for 5-7h, and then the heating is stopped and cooled to room temperature to obtain a pyridineamide bis-Schiff base alcohol solution.

[0030] In some embodiments, the alcohol solvent includes one or more of methanol, ethanol, and isopropanol.

[0031] In some embodiments, the reducing agent is NaBH 4 , NaBH 3 CN and Na(OAc) 3 One or more of BH; the molar ratio of the reducing agent to the pyridineamide bis-Schiff base is (3~5):1.

[0032] In the technical solution of the embodiment of the present application, the synthesis reaction equation of the fluorescent molecular probe is as follows: .

[0033] In some embodiments, in step S2, the pyridineamide bis-Schiff base alcohol solution is cooled to below 20° C. using an ice-water bath or an ice-salt bath, the reducing agent is dispersed in methanol or ethanol to obtain a reducing agent solution, the reducing agent solution is added to the cooled pyridineamide bis-Schiff base alcohol solution, the reaction is stirred for more than 12 hours, the solvent is evaporated, water is added to stir the reaction, and then the crude product of pyridineamide bis-Schiff base secondary amine is obtained after extraction, washing, and drying to remove the solvent.

[0034] In some embodiments, the crude product of pyridineamide bis-Schiff base secondary amine is recrystallized using ethanol, and the precipitated crystals are filtered to obtain a fluorescent molecular probe for copper ion detection.

[0035] In a third aspect, the present invention provides a method for detecting copper ions using a fluorescent molecular probe. 2+ Conduct qualitative or quantitative analysis.

[0036] In some embodiments, a fluorescent molecular probe for copper ion detection is used to detect Cu 2+ The qualitative analysis includes the following steps: Dispersing the fluorescent molecular probe in deionized water to obtain a fluorescent molecular probe solution, wherein the concentration of the fluorescent molecular probe is 7.5-10 μmol / L; The fluorescent molecular probe and the sample to be tested are mixed evenly, the metal ion concentration in the sample to be tested is more than twice the molar concentration of the fluorescent probe, and the mixture is reacted for more than 5 minutes to obtain a sample solution; The fluorescence intensity A1 of the fluorescent molecular probe solution is detected by a fluorescence spectrometer at an emission wavelength of 310 nm, and then the fluorescence intensity A2 of the sample solution is detected. If A2 is greater than or equal to 7A1, it can be determined that the sample contains Cu. 2+ Otherwise, it is determined that the sample does not contain Cu. 2+ .

[0037] In some embodiments, the fluorescent molecular probe is used to detect Cu 2+ The quantitative analysis includes the following steps: Prepare the gradient concentration of Cu 2+ Solution, Cu 2+ Fluorescent probes were added to the solution to detect the 2+ The fluorescence intensity of the solution at an emission wavelength of 310 nm was used to construct a relationship between the fluorescence intensity and Cu 2+ Standard curve of concentration; The fluorescent molecular probe and the sample to be tested are mixed evenly and reacted for more than 5 minutes to obtain a sample solution; The fluorescence intensity of the sample solution at an emission wavelength of 310 nm was detected. 2+ The concentration of Cu in the sample solution was calculated using the standard curve of concentration changes. 2+ Content; Cu 2+ The detection limit was 2.69×10 -8 mol / L.

[0038] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

[0039] 1. Preparation method Example 1 Synthesis of pyridineamide-based bis-Schiff base secondary amine fluorescent molecular probe for copper ion detection: 1) 4 mmol of intermediate N,N'-bis(2-aminoethyl)-2,6-pyridinedicarboxamide I was added to 40 mL of ethanol. a After dissolution, add dropwise into 40 mL ethanol solution containing 8.8 mmol salicylaldehyde at a rate of 5-6 s / d under magnetic stirring. After heating under reflux for about 5 h, stop heating and cool to room temperature.

[0040] 2) Use ice water / salt bath to control the temperature below 20°C, dissolve 3 times the molar number of NaBH 4 Place 20 mL of ethanol solution in a constant pressure funnel and slowly add it to the cooled reaction solution. After the addition is complete, continue to stir the reaction for 12 hours, evaporate most of the solvent at normal pressure and reduce the pressure to dryness, then add 60 mL of distilled water and stir the reaction for 10 minutes, then extract the reaction solution three times with 30 mL of ethyl acetate, combine the extracts; wash three times with 30 mL of water, combine the organic layers, and dry over anhydrous sodium sulfate.

[0041] 3) The solvent was evaporated to obtain a crude product, which was recrystallized with 20 mL of anhydrous ethanol to obtain a pure product, namely the target product, amide-based bis-Schiff base secondary amine fluorescent molecular probe, with a yield of 69%.

[0042] The infrared spectrum, mass spectrum, elemental analysis and the fluorescence performance of the pyridinamide bis-Schiff base secondary amine fluorescence-enhanced molecular probe for copper ion detection prepared in Example 1 in deuterated dimethyl sulfoxide (DMSO) solvent were analyzed. 1 H NMR, 13 C NMR spectrum data are as follows: IR(KBr / cm -1 ):3287(υH 2O / OH),1663(υamideI),1604(υN-H),1398(υC-N),1541(υamideII),1256(υphenylC-OH),1109,1460(υpyridyl);UV-Vis(λmax / nm)(ε):272(7137);Fluorescence: λEx (305), λEm (310), I (6.71%). 1 H NMR (DMSO-d 6 )δ:4.85(s, H,amide), 8.32(s, H, amide), 3.75(m, 8H, -NH–CH 2 –CH 2 –NH), 2.98 (t, J = 6.7 Hz,4H, N–CH 2 –Ph), 7.25(m, 3H, pyridyl H), 8.89 (s, OH, 2H), 6.89(m, 8H, phenylH), 4.85 (s, 2 HC–NH). 13 EI-MSm / Z (%): 464.3 (M+H) + , calculated: 464.2. The calculated values ​​of the probe Lin elementary analysis (%): C 64.79, H 6.26, N 15.12%; the measured values ​​(%): C 65.04, H 6.41, N 15.32%.

[0043] From the above data, it can be seen that the structure of the pyridinamide bis-Schiff base secondary amine fluorescence-enhanced molecular probe for copper ion detection prepared in this example is as follows: .

[0044] Example 2 The fluorescence emission spectrum of the pyridine amide bis-Schiff base secondary amine fluorescent molecular probe prepared in Example 1 was tested as follows: the fluorescent molecular probe L was added to deionized water at a concentration of 10 μmol / L, reacted for 5 minutes and fixed to volume, and its fluorescence emission spectrum was detected. Figure 1 As shown. Figure 1 It can be seen that the fluorescent molecular probe L prepared in Example 1 has an emission peak at 310 nm and a strong fluorescence intensity.

[0045] Example 3 The pyridine amide-based bis-Schiff base secondary amine fluorescent molecular probe prepared in Example 1 was tested for its affinity to Cu 2+ The fluorescence emission spectrum after the action is as follows: The test temperature of the fluorescence spectrum was 25°C and the test condition was a deionized water buffer system. Before the test, copper nitrate was prepared into 1×10 -3 mol / L stock solution. When testing, pipette 100uL of a 1×10 -3 mol / L probe molecule L solution into a series of 10mL volumetric flasks, and then transfer 1 times the molar number of the corresponding Cu 2+ Solution, first add 1mL of deionized water, react for 5 minutes, and dilute to 1×10 -5 mol / L, and then the fluorescence emission spectrum was tested after sufficient mixing. The obtained fluorescence emission spectrum is shown in Figure 2 ,Depend on Figure 2 It can be seen that when Cu is added 2+ When the fluorescence intensity increases by 7 times, the fluorescence intensity increases by 7 times. 2+ Ions have good selectivity and recognition ability.

[0046] Embodiments 4 to 10 Detection of the pyridine amide-based bis-Schiff base secondary amine fluorescent molecular probe prepared in Example 1 on Cu 2+ Selective sensitivity, the specific method is as follows: According to the concentration of fluorescent molecular probe L of 10 μmol / L, the fluorescent molecular probe was added to deionized water, and 2 μmol / L, 5 μmol / L, 8 μmol / L, 10 μmol / L, 20 μmol / L, 30 μmol / L, and 40 μmol / L of Cu were added respectively. 2+ , react for 5 minutes and fix the volume, test the change curve of fluorescence emission spectrum, see Figure 3 .from Figure 3 It can be seen from the image that the fluorescence intensity of the probe increases with the Cu 2+ As the concentration of Cu increases, the fluorescence intensity increases continuously, and the fluorescence intensity is similar to that of Cu 2+ The concentration change shows a good linear relationship.2+ When the concentration increased to 4 equivalents (40 μmol / L), the fluorescence intensity almost stopped changing and reached the highest level.

[0047] Figure 4 The fluorescent molecular probes according to Examples 4 to 10 detect Cu 2+ The detection limit test curve diagram, the horizontal axis is Cu 2+ The concentration C is in μmol / L, the ordinate is the fluorescence intensity F, and the fitted linear equation is: F=25.52429+255.53628C; the correlation coefficient is R 2 = 0.99569, SD = 2.29183. The formula for calculating the limit of detection is LOD = 3σ / K, where σ is the standard deviation SD and K is the slope of the fitting line. By calculation, we can get the fluorescence molecular probe L for Cu 2+ The detection limit was 2.69×10 -8 mol / L.

[0048] Figure 5 is the fluorescence intensity and the corresponding Cu measured in Examples 4 to 10 2+ Benesi-Hildebrand linear analysis curve obtained by concentration, 1 / C ( Cu 2+ ) as the horizontal axis and 1 / ( F - F0) as the vertical axis, the fitted linear equation is: F =-1.52363E-4 + 0.00779x; According to the fluorescence titration data, the BenesiHildebrand equation based on 1:1 was used, and the fitting result was a straight line, indicating that the probe L and Cu 2+ The complexation ratio is 1:1, and the R 2 is 0.9951, and the complex constant is the inverse of the slope of the fitting line. The complex constant K is calculated to be 1.28×10 8 ( mol / L) -1 .

[0049] Embodiments 11 to 17 Detection of the pyridine amide-based bis-Schiff base secondary amine fluorescent molecular probe prepared in Example 1 and Cu 2+ Fluorescence emission diagram in solutions with different pH values, the specific method is as follows: The fluorescent molecular probe L was added into deionized water at a concentration of 10 μmol / L. In Examples 11 to 17, the pH of the fluorescent molecular probe L solution was adjusted to 2, 3, 4, 5, 6, 6.5, 7, 7.5, and 8 using acid or alkali solution, respectively, and the fluorescence intensity A1 at an emission wavelength of 310 nm was detected.

[0050] Then, according to the concentration of fluorescent molecular probe L of 10 μmol / L, the fluorescent molecular probe was added to deionized water, and 10 μmol / L Cu 2+ In Examples 11 to 17, the fluorescent molecular probe L+Cu 2+ The pH value of the solution was adjusted to 2, 3, 4, 5, 6, 6.5, 7, 7.5, and 8, and the solution was reacted for 5 minutes and fixed to volume, and the fluorescence intensity A2 was detected at an emission wavelength of 310 nm.

[0051] Fluorescent molecular probes and Cu 2+ The fluorescence emission diagrams in solutions with different pH values ​​are shown in Figure 6 ,from Figure 6 It can be seen that the fluorescent molecular probe L and Cu 2+ The optimal detection pH range is 6.5~7.5.

[0052] Embodiment 18 The pyridine amide-based bis-Schiff base secondary amine fluorescent molecular probe prepared in Example 1 was tested for its affinity to Cu 2+ The fluorescence emission spectra corresponding to different times are as follows: The test temperature of the fluorescence spectrum was 25 °C, and the test conditions were deionized water buffer system. Before the test, copper nitrate was prepared into 1×10 -3 mol / L stock solution, when testing, pipette 100uL of a concentration of 1×10 -3 mol / L probe molecule L solution into a series of 10mL volumetric flasks, and then transfer 1 times the molar number of the corresponding Cu 2+ Solution, first add 1mL of deionized water to make the volume 1×10 -5 mol / L, fluorescence emission spectrum test was performed every 30s, and the test response time was 0~16min.

[0053] Fluorescent molecular probes and Cu 2+ The corresponding fluorescence emission spectra at different times are shown in Figure 7 ,from Figure 7 It can be seen that the fluorescent molecular probe L and Cu 2+ The response time is 5 minutes.

[0054] Embodiment 19 The tap water sample was mixed with the anhydrous ethanol stock solution of the pyridine amide-based bis-Schiff base secondary amine fluorescent molecular probe prepared in Example 1 (1.00×10 -4 mol·L -1 ) were mixed in a volume ratio of 1:19, reacted for 5 minutes, and the fluorescence intensity was detected at an emission wavelength of 310 nm.

[0055] Embodiment 20 The tap water sample was mixed with the anhydrous ethanol stock solution of the pyridine amide bis-Schiff base secondary amine fluorescent molecular probe prepared in Example 1 (1.00×10 -4 mol·L -1 ) were mixed in a volume ratio of 1:19, and 10 μmol Cu was added thereto 2+ , react for 5 minutes, and detect the fluorescence intensity at an emission wavelength of 310 nm.

[0056] Embodiment 21 The difference between Example 21 and Example 19 is that the water sample is water from Huangjia Lake River in Wuhan City.

[0057] Embodiment 22 The difference between Example 22 and Example 20 is that the water sample is water from Huangjia Lake River in Wuhan City.

[0058] The Cu of Examples 19 to 22 was calculated according to the linear equation obtained in Examples 4 to 10. 2+ Detection value, and calculate the recovery rate = (Cu 2+ Detection value / Cu 2+ Addition amount) × 100%, the test results are shown in Table 1 below.

[0059] Table 1 Cu in actual water samples in Examples 19 to 22 2+ Recovery test

[0060] As can be seen from Table 1, the fluorescent molecular probe L cannot detect Cu in these two actual water samples. 2+ This is because the Cu 2+ The content is lower than the detection limit of this method; the fluorescent probe L prepared in Example 1 is used to detect Cu 2+ The test results are consistent with the actual amount of addition, and the recovery rate is high. Therefore, the fluorescent probe L can be applied to the actual water sample 2.69×10 -8 ~40×10 -6 Cu in mol / L concentration range 2+ Detection.

[0061] The Schiff base designed by Chen Jia's research group in the paper "Synthesis and Recognition Performance of Copper Ion Fluorescent Probe" 2+ The detection limit is 0.15 μmol / L, and the detection range is 0.2~2 μmol / L. The probe designed and synthesized by Wang Yuting and Zhang Yan's research group in "Synthesis of a tetraphenylethylene fluorescent probe and its application in copper ion detection" is very sensitive to Cu 2+The detection range is between 0.05 and 8 μmol / L, and the detection limit is 25 nmol / L. Compared with the above copper ion probes, the probe in this application has a wide detection range and a low detection limit.

[0062] Comparative Examples 1 to 10 The fluorescence emission spectra of the pyridineamide-based bis-Schiff base secondary amine fluorescent molecular probe prepared in Example 1 after reacting with different metal ions are tested. The specific method is as follows: The test temperature of the fluorescence spectrum was 25 °C, and the test conditions were deionized water buffer system. Before the test, the metal ion nitrate was prepared into 1×10 -3 mol / L stock solution, the metal ions in comparative examples 1 to 10 are K + Mg 2+ , Ca 2+ , Mn 2+ 、Zn 2+ , Pb 2+ , Hg 2+ 、Cd 2+ 、Co 2+ Cr 3+ During the test, 100uL of 1×10 -3 mol / L probe molecule L solution into a series of 10mL volumetric flasks, and then transfer 1 times the mole of the corresponding metal ion (K + Mg 2+ , Ca 2+ , Mn 2+ 、Zn 2+ , Pb 2+ , Hg 2+ 、Cd 2+ 、Co 2+ Cr 3+ ) solution, add 1 mL of deionized water, react for 5 minutes, and dilute to 1 × 10 -5 mol / L, and then the fluorescence emission spectrum test was performed after thorough mixing.

[0063] The obtained fluorescence emission spectrum is shown in Figure 2 ,Depend on Figure 2 It can be seen that adding Cu 2+ When different metal ions other than Cu are added, the characteristic emission peaks do not change significantly. 2+ When the fluorescence intensity increases by 7 times, the fluorescence intensity increases by 7 times. 2+ Ions have good selectivity and recognition ability.

[0064] Comparative Examples 11-21 The fluorescent molecular probe prepared in Example 1 was tested for its resistance to metal ion interference. The method was as follows: first, add 2 times the amount of Cu2+ Then, add Cu 2+ The concentration of K + Mg 2+ , Ca 2+ , Mn 2+ , Cu 2+ 、Zn 2+ , Pb 2+ , Hg 2+ 、Cd 2+ 、Co 2+ Cr 3+ Coexist metal ions, mix well and then add probe molecule L at a concentration of 10μmol / L, react for 5 minutes and fix the volume, then measure its fluorescence intensity.

[0065] The obtained fluorescence emission spectrum is shown in Figure 8 ,Depend on Figure 8 It can be seen that other common metal ions (Ca 2+ ,Co 2+ , K + ,Mg 2 + , Hg 2+ ) and Cu 2+ At the same time, Cu 2+ It can still greatly enhance the fluorescence intensity of the fluorescent molecular probe, but Cr 3+ The presence of ions significantly reduces the fluorescence intensity of the system, which has a certain impact on the accuracy of the detection results.

[0066] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A fluorescent molecular probe for copper ion detection, characterized in that: The fluorescent molecular probe structural formula for copper ion detection is: 。 2. The method for preparing a fluorescent molecular probe for copper ion detection according to claim 1, characterized in that: The steps include: (1) Salicylaldehyde reacts with N,N'-bis(2-aminoethyl)-2,6-pyridinedicarboxamide to obtain pyridineamide bis-Schiff base; (2) The pyridineamide bis-Schiff base is reduced using a reducing agent to obtain a fluorescent molecular probe for copper ion detection.

3. The method for preparing a fluorescent molecular probe for copper ion detection according to claim 2, characterized in that: In step S1, salicylaldehyde and N,N'-bis(2-aminoethyl)-2,6-pyridinedicarboxamide are added to an alcohol solvent, heated to reflux at 65-80°C, reacted for 5-7 hours, and then the heating is stopped and cooled to room temperature to obtain a pyridineamide bis-Schiff base alcohol solution.

4. The method for preparing a fluorescent molecular probe for copper ion detection according to claim 3, characterized in that: The alcohol solvent includes one or more of methanol, ethanol and isopropanol.

5. The method for preparing a fluorescent molecular probe for copper ion detection according to claim 3, characterized in that: The reducing agent in step S2 is one or more of NaBH4, NaBH3CN and Na(OAc)3BH; the molar ratio of the reducing agent to the pyridineamide bis-Schiff base is (3-5):

1.

6. The method for preparing a fluorescent molecular probe for copper ion detection according to claim 3, characterized in that: In step S2, the pyridinamide bis-Schiff base alcohol solution is cooled to below 20° C. by using an ice-water bath or an ice-salt bath, a reducing agent is dispersed in methanol or ethanol to obtain a reducing agent solution, and the reducing agent solution is added to the cooled pyridinamide bis-Schiff base alcohol solution, stirred to react for more than 12 hours, the solvent is evaporated, water is added to stir the reaction, and then the crude pyridinamide bis-Schiff base secondary amine product is obtained after extraction, washing, drying, and removal of the solvent.

7. The method for preparing a fluorescent molecular probe for copper ion detection according to claim 6, characterized in that: The crude product of the pyridineamide bis-Schiff base secondary amine is recrystallized by ethanol, and the precipitated crystals are filtered to obtain a fluorescent molecular probe for copper ion detection.

8. A use of a fluorescent molecular probe for copper ion detection, characterized in that: The fluorescent molecular probe for copper ion detection described in claim 1 is used to detect Cu 2+ Conduct qualitative or quantitative analysis.

9. The use of the fluorescent molecular probe for copper ion detection according to claim 8, characterized in that: The fluorescent molecular probe for copper ion detection is used to detect Cu 2+ The qualitative analysis includes the following steps: Dispersing the fluorescent molecular probe in deionized water to obtain a fluorescent molecular probe solution, wherein the concentration of the fluorescent molecular probe is 7.5-10 μmol / L; The fluorescent molecular probe and the sample to be tested are mixed evenly, the metal ion concentration in the sample to be tested is more than twice the molar concentration of the fluorescent probe, and the mixture is reacted for more than 5 minutes to obtain a sample solution; The fluorescence intensity A1 of the fluorescent molecular probe solution is detected by a fluorescence spectrometer at an emission wavelength of 310 nm, and then the fluorescence intensity A2 of the sample solution is detected. If A2 is greater than or equal to 7A1, it can be determined that the sample contains Cu. 2+ Otherwise, it is determined that the sample does not contain Cu. 2+ .

10. The use of the fluorescent molecular probe for copper ion detection according to claim 8, characterized in that: The fluorescent molecular probe for copper ion detection is used to detect Cu 2+ The quantitative analysis includes the following steps: Prepare the gradient concentration of Cu 2+ Solution, Cu 2+ Fluorescent probes were added to the solution to detect the 2+ The fluorescence intensity of the solution at an emission wavelength of 310 nm was used to construct a relationship between the fluorescence intensity and Cu 2+ Standard curve of concentration; The fluorescent molecular probe and the sample to be tested are mixed evenly and reacted for more than 5 minutes to obtain a sample solution; The fluorescence intensity of the sample solution at an emission wavelength of 310 nm was detected, and the Cu content in the sample solution was calculated according to the standard curve. 2+ Content; The Cu 2+ The detection limit was 2.69×10 -8 mol / L.

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