Copper ion fluorescence probe, copper ion detection test paper, preparation method of copper ion fluorescence probe, copper ion detection test paper, kit and application of kit

Abstract

The invention relates to the technical field of copper ion detection, and discloses a copper ion fluorescent probe, copper ion detection test paper, a preparation method of the copper ion fluorescent probe, a kit and application of the kit, the structural formula of the copper ion fluorescent probe is # imgabs0 #, and R is an electron withdrawing group containing active methyl. A kit prepared on the basis of the copper ion fluorescent probe is good in portability and low in cost, professional personnel do not need to use professional equipment, and qualitative or semi-quantitative real-time rapid detection can be conducted on copper ions under the assistance of an ultraviolet detection lamp.

Description

Technical Field

[0001] The present invention relates to the technical field of copper ion detection, and particularly to a copper ion fluorescent probe, a copper ion detection test strip, a preparation method thereof, a kit, and an application of the kit. Background Art

[0002] Copper ion (Cu 2+ ) is a heavy metal element widely distributed in nature. To avoid heavy metal poisoning, the use of copper ions in food is strictly restricted. However, some merchants still use copper sulfate for its coloring effect in Chinese herbal medicines and its bactericidal and fresh-keeping effect in vegetables, which easily leads to serious over-standard copper ion residues in Chinese herbal medicine products and vegetables, causing serious health and safety and environmental problems. In addition, excessive enrichment of copper ions in water will also cause great harm to the surrounding environment.

[0003] Copper ions seriously affect food and environmental safety. To increase the detection of copper ions in food and the environment, rapid detection and analysis of copper ions are required. However, existing copper ion detection requires professionals to use professional equipment, and the detection process is relatively complex, costly, and the users and sites are restricted. Therefore, how to achieve rapid on-site detection of copper ions at low cost is an urgent problem to be solved. Summary of the Invention

[0004] In view of this, the present invention provides a copper ion fluorescent probe, a copper ion detection test strip, a preparation method thereof, a kit, and an application of the kit to solve the problem that the existing copper ion detection process cannot achieve rapid on-site detection due to complex detection process, high cost, and high professionalism.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a copper ion fluorescent probe, and the structural formula of the copper ion fluorescent probe is:

[0007]

[0008] wherein R is an electron-withdrawing group containing an active methyl group.

[0009] Preferably, the R is selected from the following groups:

[0010]

[0011] In the second aspect, the present invention provides a preparation method of the copper ion fluorescent probe according to any one of the above, comprising the following steps:

[0012] In a first organic solvent, 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and compound R are mixed, and a catalyst is added and the mixture is refluxed with condensation to carry out the Knoevenagel condensation reaction to obtain a copper ion fluorescent probe;

[0013] The compound R is an organic compound containing an active methyl group.

[0014] Preferably, the molar ratio of 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde to the compound R is 1:1 - 1.2.

[0015] Preferably, the ratio of the sum of the molar amounts of 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and compound R to the molar amount of the catalyst is 1:n, where n ≥ 3.

[0016] Preferably, the first organic solvent is ethanol.

[0017] Preferably, the catalyst is selected from piperidine and pyridine.

[0018] Preferably, the temperature of the reflux with condensation is 80 - 90 °C and the time is 12 - 14 h.

[0019] Preferably, the compound R is selected from the following structures:

[0020]

[0021] In a third aspect, the present invention provides a copper ion detection test strip, comprising a reaction medium paper and a copper ion fluorescent probe loaded on the reaction medium paper, wherein the copper ion fluorescent probe is the above copper ion fluorescent probe or the copper ion fluorescent probe prepared by the method described in any one of the above.

[0022] In a fourth aspect, the present invention provides a method for preparing a copper ion detection test strip, comprising the following steps: soaking the reaction medium paper in the copper ion fluorescent probe solution and then drying to obtain a copper ion detection test strip.

[0023] Preferably, the copper ion fluorescent probe solution is obtained by dissolving the copper ion fluorescent probe in a second organic solvent.

[0024] Preferably, the concentration of the copper ion fluorescent probe solution is 0.1 - 0.3 mmol / L.

[0025] Preferably, the second organic solvent includes one or more of methanol, ethanol, acetone, and tetrahydrofuran.

[0026] Preferably, the soaking time is 5 - 10 min.

[0027] Fifth aspect, the present invention provides a kit, which includes a copper ion fluorescent probe, a reaction medium paper, a solvent and an ultraviolet detection lamp, wherein the copper ion fluorescent probe is the above-mentioned copper ion fluorescent probe or the copper ion fluorescent probe prepared by the method described in any one of the above, and the solvent is used to dissolve the copper ion fluorescent probe;

[0028] Or the kit includes a copper ion test paper, a fluorescence color comparison card and an ultraviolet detection lamp, wherein the copper ion test paper is the above-mentioned copper ion test paper or the copper ion test paper prepared by the method described in any one of the above.

[0029] Sixth aspect, the present invention provides an application of the above-mentioned kit, and the kit is used for detecting copper ions in food materials, medicinal materials or water environment.

[0030] The present invention provides a copper ion fluorescent probe, a copper ion test paper, a preparation method thereof, a kit and an application of the kit. Compared with the prior art, the beneficial effects are as follows:

[0031] The kit prepared based on the copper ion fluorescent probe of the present invention has good portability and low cost. Without the need for professionals to use professional equipment, it can qualitatively or semi-quantitatively detect copper ions in real time and quickly with the assistance of an ultraviolet detection lamp. Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0033] Figure 1 It is the high-resolution mass spectrum of the copper ion fluorescent probe ZYC in Example 1 of the present invention;

[0034] Figure 2 It is the 1 HNMR spectrum of the copper ion fluorescent probe ZYC in Example 1 of the present invention;

[0035] Figure 3 It is the 13 C NMR spectrum of the copper ion fluorescent probe ZYC in Example 1 of the present invention;

[0036] Figure 4 It is the high-resolution mass spectrum of the copper ion fluorescent probe VIC in Example 2 of the present invention;

[0037] Figure 51H NMR spectrum of the copper ion fluorescent probe VIC of Example 2 of the present invention 1 HNMR chart

[0038] Figure 6 13C NMR spectrum of the copper ion fluorescent probe VIC of Example 2 of the present invention 13 CNMR chart

[0039] Figure 7 High resolution mass spectrum of the copper ion fluorescent probe QYC of Example 3 of the present invention

[0040] Figure 8 1H NMR spectrum of the copper ion fluorescent probe QYC of Example 3 of the present invention 1 HNMR chart

[0041] Figure 9 13C NMR spectrum of the copper ion fluorescent probe QYC of Example 3 of the present invention 13 CNMR chart

[0042] Figure 10 Ultraviolet absorption spectrum of the copper ion fluorescent probe ZYC (A), ultraviolet-visible absorption spectrum of the copper ion fluorescent probe ZYC in the presence of various metal ions (B);

[0043] Figure 11 Fluorescence spectrum of the copper ion fluorescent probe ZYC (A), fluorescence spectrum of the copper ion fluorescent probe ZYC in the presence of various metal ions (B), fluorescence response of the copper ion fluorescent probe ZYC to copper ions in the presence of different metal ions (C), fluorescence stability curve of the copper ion fluorescent probe ZYC (D);

[0044] Figure 12 Schematic diagram of the influence of pH value on the fluorescence response of the copper ion fluorescent probe ZYC (A), response time graph of the copper ion fluorescent probe ZYC to different concentrations of copper ions (B), schematic diagram of the influence of copper ion concentration on the fluorescence intensity of the copper ion fluorescent probe ZYC (C);

[0045] Figure 13 High resolution mass spectrum of the copper ion fluorescent probe ZYC in the presence of copper ions (A), 1 H NMR titration test result graph (B);

[0046] Figure 14 Job's curve of the copper ion fluorescent probe ZYC for recognizing copper ions (A), standard curve of the complexation constant of the copper ion fluorescent probe ZYC after recognizing copper ions (B), standard curve of the quenching constant of the copper ion fluorescent probe ZYC after recognizing copper ions (C);

[0047] Figure 15 Schematic diagram of the kit

[0048] Figure 16 Schematic diagrams of a colorimetric card and a fluorescence colorimetric card (A), schematic diagram of using a smartphone to extract the colors of the colorimetric card and the fluorescence colorimetric card (B), calibration curve of the colorimetric card for copper ion concentration (C), calibration curve of the fluorescence colorimetric card for copper ion concentration (D), schematic diagram of the process of detecting copper ion concentration using a test kit (E);

[0049] Figure 17 Schematic diagram of the detection of excessive copper ions in senna leaves (A), zongzi leaves (B), Chinese chives (C), and garlic bolts (D) by a copper ion test strip. Detailed implementation manners

[0050] The present invention will be described below through specific examples. Those skilled in the art can understand that the following specific examples are only for the purpose of illustration and do not limit the scope of the present invention in any way. In addition, in the following examples, unless otherwise specified, the reagents and equipment used are commercially available. If the specific processing conditions and methods are not clearly described in the following examples, the conditions and methods known in the art can be used for processing.

[0051] In the first aspect of the present invention, a copper ion fluorescent probe is proposed, and the structural formula of the copper ion fluorescent probe is:

[0052]

[0053] wherein, R is an electron-withdrawing group containing an active methyl group.

[0054] In some embodiments of the present invention, the R is selected from the following groups:

[0055]

[0056] In the second aspect of the present invention, a preparation method of the copper ion fluorescent probe according to any one of the above is proposed, including the following steps:

[0057] In a first organic solvent, 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and compound R are mixed, and a catalyst is added for reflux condensation to carry out the Knoevenagel condensation reaction to obtain the copper ion fluorescent probe;

[0058] The compound R is an organic compound containing an active methyl group.

[0059] In some embodiments of the present invention, the molar ratio of 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde to the compound R is 1:1 - 1.2, for example, it can be 1:1, 1:1.05, 1:1.1, 1:1.2, etc.

[0060] In some embodiments of the present invention, the preparation method of 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde comprises the following steps: Dissolve 3-chloromethyl-5-nitrosalicylaldehyde and diethanolamine in tetrahydrofuran, add triethylamine for reaction, then carry out reflux condensation. After the reaction is completed, cool to room temperature, and filter to obtain an orange-red solid, which is 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde.

[0061] Among them, the molar ratio of 3-chloromethyl-5-nitrosalicylaldehyde to diethanolamine is 1:1 - 1.2, for example, it can be 1:1, 1:1.1, 1:1.2, etc.; the molar ratio of 3-chloromethyl-5-nitrosalicylaldehyde to triethylamine is 1:3 - 4, for example, it can be 1:3, 1:3.5, 1:4, etc.; the temperature of the reaction is 0 °C, the reaction time is 50 - 70 min, for example, it can be 50 min, 60 min, 70 min, etc.; the temperature of the reflux condensation is greater than the solvent temperature, and the reflux condensation time is 15 - 17 h, for example, it can be 15 h, 16 h, 17 h, etc.; the filtration can be suction filtration, and no special limitation is imposed on this, as long as the solid product can be obtained.

[0062] In some embodiments of the present invention, the first organic solvent is ethanol, and no special limitation is imposed on the addition amount of the first organic solvent, as long as 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and compound R are completely dissolved.

[0063] In some embodiments of the present invention, the catalyst is selected from one of piperidine and pyridine, preferably piperidine; the ratio of the sum of the molar amounts of 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and compound R to the molar amount of the catalyst is 1:n, where n ≥ 3, for example, it can be 1:3, 1:4, 1:5, etc.

[0064] In some embodiments of the present invention, the temperature of the reflux condensation is 80 - 90 °C, for example, it can be 80 °C, 85 °C, 90 °C, etc., and the reflux condensation time is 12 - 14 h, for example, it can be 12 h, 13 h, 14 h, etc.

[0065] In some embodiments of the present invention, the preparation method of the copper ion fluorescent probe further comprises the following steps: After the reaction is completed, cool to room temperature, filter and dry to obtain the copper ion fluorescent probe in solid state.

[0066] In some embodiments of the present invention, the drying time is 24 - 30 h. For example, it can be 24 h, 26 h, 28 h, 30 h, etc. There is no special limitation on the drying method. For example, it can be air drying at room temperature or drying using a general drying oven; the filtration method can be suction filtration. There is no special limitation on the filtration method, and it can be adjusted according to the actual situation.

[0067] In some embodiments of the present invention, the compound R is selected from the following structures:

[0068]

[0069] In the third aspect of the present invention, a copper ion detection test strip is proposed. The copper ion detection test strip includes a reaction medium paper and a copper ion fluorescent probe loaded on the reaction medium paper, wherein the copper ion fluorescent probe is the above-mentioned copper ion fluorescent probe or the copper ion fluorescent probe prepared by the method described in any one of the above.

[0070] In some embodiments of the present invention, the reaction medium paper can be a filter paper commonly used in laboratories, and there is no special limitation on the pore size of the filter paper. In a specific embodiment of the present invention, a filter paper of the Hoffman brand is used.

[0071] In the fourth aspect of the present invention, a method for preparing a copper ion detection test strip is proposed, which includes the following steps: soaking the reaction medium paper in the copper ion fluorescent probe solution and then drying to obtain the copper ion detection test strip.

[0072] In some embodiments of the present invention, the copper ion fluorescent probe solution is obtained by dissolving the copper ion fluorescent probe in a second organic solvent. The concentration of the copper ion fluorescent probe solution is 0.1 - 0.3 mmol / L, specifically referring to the concentration of the copper ion fluorescent probe in the solution. The concentration can be, for example, 0.1 mmol / L, 0.2 mmol / L, 0.3 mmol / L, etc.; the second organic solvent is a volatile organic solvent, such as methanol, ethanol, acetone, tetrahydrofuran, etc.

[0073] In some embodiments of the present invention, the soaking time is 5 - 10 min. For example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0074] In the fifth aspect of the present invention, a kit is proposed.

[0075] In some embodiments of the present invention, the kit includes a copper ion fluorescent probe, a reaction medium paper, a solvent, and an ultraviolet detection lamp, wherein the copper ion fluorescent probe is the above-mentioned copper ion fluorescent probe or the copper ion fluorescent probe prepared by the method described in any one of the above, and the solvent is used to dissolve the copper ion fluorescent probe, that is, the second organic solvent described above.

[0076] In some other embodiments of the present invention, the kit includes a copper ion test strip, a fluorescence color comparison card, and an ultraviolet detection lamp, wherein the copper ion test strip is the above-mentioned copper ion test strip or the copper ion test strip prepared by the method described in any one of the above.

[0077] It should be noted that the main difference between the above two kits lies in whether they include a fluorescence color comparison card. Compared with the scheme including a fluorescence color comparison card, the scheme without a fluorescence color comparison card can prepare a fluorescence color comparison card and / or a copper ion concentration standard curve card according to the range of copper ion concentrations to be detected as needed, with better flexibility, and more accurate copper ion concentration detection can be achieved according to the copper ion concentration standard curve card.

[0078] In the scheme without a fluorescence color comparison card, when using the kit, a fluorescence color comparison card can be prepared by the following steps. The preparation method of the fluorescence color comparison card is as follows: First, take a copper ion fluorescent probe, a reaction medium paper, and a solvent, and prepare a copper ion test strip by the method described above; then soak multiple copper ion test strips in a series of copper ion solutions with different concentrations respectively, and after taking them out, obtain a fluorescence color comparison card according to the colors of the copper ion test strips under ultraviolet light.

[0079] In some embodiments of the present invention, the copper ion test strip is soaked in a series of copper ion solutions with different concentrations. The soaking time is 5 - 10 min, for example, it can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.; the concentrations of the series of copper ion solutions can be adjusted according to the actual situation, and no special limitation is imposed thereon. For example, the concentrations of the prepared series of copper ion solutions are 0, 15 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, and 80 μmol / L respectively. The colors of each copper ion test strip are obtained under ultraviolet light, and a fluorescence color comparison card is prepared.

[0080] In some embodiments of the present invention, the R / (G + B) value of the test strip is extracted from the fluorescence color comparison card by using the color recognition software of a smart phone to obtain a copper ion concentration calibration curve, and a copper ion concentration standard curve card can be prepared.

[0081] It should be noted that the wavelength of the ultraviolet light of the ultraviolet detection lamp described in the present invention is 365 nm.

[0082] In some embodiments of the present invention, a copper ion concentration standard curve card can also be placed in the kit to achieve more accurate detection of copper ion concentration.

[0083] In the fifth aspect of the present invention, the present invention provides an application of the above-mentioned kit, and the kit is used for detecting copper ions in food materials, medicinal materials or water environment.

[0084] It can be understood that when detecting copper ions in food materials and medicinal materials, after spraying water on the surfaces of the food materials and medicinal materials, wiping the surfaces with a copper ion test paper completes the sampling; while when detecting copper ions in a water sample, dropping the well-mixed water sample on the copper ion test paper and standing still completes the sampling. The standing time is generally within 1 minute, and no special limitation is imposed on this.

[0085] As Figure 16 (E) shows, if it is necessary to use the kit to qualitatively or semi-quantitatively detect the copper ion concentration, under the irradiation of 365 nm ultraviolet light, comparing the copper ion test paper after sampling with the fluorescence colorimetric card can achieve this; if it is necessary to use the kit to accurately quantitatively detect the copper ion concentration, it is necessary to use an ultraviolet dark box and a smart phone to extract the R / (G + B) value of the copper ion test paper after sampling, and then read the value with the copper ion concentration standard curve card.

[0086] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with specific embodiments. The embodiments of this application are only for illustration. All other embodiments obtained by those skilled in the art without creative efforts fall within the scope of protection of the present invention.

[0087] Example 1

[0088] This example provides a preparation method of a copper ion fluorescent probe ZYC, and the specific steps are as follows:

[0089] (1) Dissolve 3-chloromethyl-5-nitrosalicylaldehyde (1.0 g, 4.64 mmol) and diethanolamine (0.49 g, 4.66 mmol) in 50 mL of tetrahydrofuran, add 2 mL of triethylamine (1.46 g, 14.39 mmol), react at 0 °C for 60 min, then reflux under condensation for 17 h. After the reaction is completed, cool to room temperature, perform suction filtration, and rotary evaporate the filtrate to obtain 1.27 g of orange-red solid, namely 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde, with a yield of 96%;

[0090] The synthesis reaction formula of 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde is as follows:

[0091]

[0092] (2) Dissolve 3 - ((bis(2 - hydroxyethyl)amino)methyl)-2 - hydroxy - 5 - nitrobenzaldehyde (0.15 g, 0.53 mmol) and compound R 2-(3 - cyano - 4,5,5 - trimethylfuran - 2(5H)-methylene)malononitrile (0.11 g, 0.55 mmol) in 15 mL of absolute ethanol, then add 0.2 mL of pyridine, reflux under condensation for 12 h, cool to room temperature, perform suction filtration, and dry at 25 °C for 24 h to obtain a dark green solid, which is the copper ion fluorescent probe ZYC with a yield of 80%.

[0093] The synthesis reaction formula of the copper ion fluorescent probe ZYC is as follows:

[0094]

[0095] As Figures 1-3 shown, they are respectively the high - resolution mass spectrum, 1 HNMR spectrum and 13 C NMR spectrum of the copper ion fluorescent probe ZYC. It can be seen from the figures that 1 H NMR (400 MHz, DMSO - d6) δ (ppm): 9.53 (s, 1H), 8.61 (d, J = 3.1 Hz, 1H), 8.37 - 8.04 (m, 2H), 7.90 (d, J = 15.8 Hz, 1H), 5.26 (s, 2H), 4.35 (s, 2H), 3.78 (t, J = 5.0 Hz, 4H), 3.22 (t, J = 4.9 Hz, 4H), 1.80 (s, 6H). 13 C NMR (101 MHz, DMSO - d6) δ (ppm): 179.1, 178.3, 177.7, 149.2, 133.6, 131.5, 130.2, 122.6, 121.8, 113.8, 113.8, 112.9, 111.9, 99.0, 56.1, 55.5, 54.5, 26.4. HR - MS (positive mode, m / z) for [ZYC + H + + , calculated value 466.1721, found: 466.1708.

[0096] Example 2

[0097] This example provides a preparation method of a copper ion fluorescent probe VIC. The preparation method of the copper ion fluorescent probe VIC is basically the same as that of Example 1, and the only difference lies in the different compound R.

[0098] The synthesis reaction formula of the copper ion fluorescent probe VIC is as follows:​

[0099]

[0100] As Figures 4-6 shown, they are respectively the high-resolution mass spectrum of the copper ion fluorescent probe VIC, 1 HNMR spectrum and 13 C NMR spectrum. As can be seen from the figures, 1 H NMR (400 MHz, DMSO-d6) δ (ppm): 8.39 (d, J = 3.0 Hz, 1H), 7.98 (d, J = 3.0 Hz, 1H), 7.69 (d, J = 16.1 Hz, 1H), 7.47 (d, J = 16.1 Hz, 1H), 6.79 (s, 1H), 5.20 (s, 2H), 4.30 (s, 2H), 3.74 (t, J = 5.3 Hz, 4H), 3.12 (t, J = 5.3 Hz, 4H), 2.56 (d, J = 28.7 Hz, 4H), 1.02 (s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ (ppm): 175.8, 170.7, 157.9, 135.5, 132.2, 128.3, 127.7, 126.4, 123.6, 121.8, 120.8, 114.7, 74.8, 57.2, 55.9, 54.4, 42.8, 38.6, 32.1, 27.9. HR-MS (positive mode, m / z) for [VIC-H + - , calculated value: 451.1987, found: 451.3005.

[0101] Example 3

[0102] This example provides a preparation method of the copper ion fluorescent probe QYC. The preparation method of the copper ion fluorescent probe QYC is basically the same as that of Example 1, and the only difference lies in the different compound R.

[0103] The synthesis reaction formula of the copper ion fluorescent probe QYC is as follows:

[0104]

[0105] As Figures 7-9 shown, they are respectively the high-resolution mass spectrum of the copper ion fluorescent probe QYC, 1 HNMR spectrum and 13 C NMR spectrum. As can be seen from the figures, 1 ​1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.04 (s, 1H), 8.90 (d, J = 9.1 Hz, 1H), 8.67 (d, J = 15.2 Hz, 1H), 8.56 - 8.47 (m, 2H), 8.44 (d, J = 9.0 Hz, 1H), 8.32 - 8.21 (m, 2H), 8.17 - 8.07 (m, 2H), 7.88 (t, J = 7.5 Hz, 1H), 5.28 (s, 2H), 4.42 (s, 3H), 4.40 (s, 2H), 3.80 (s, 4H), 3.25 (t, J = 5.0 Hz, 4H). 13 13C NMR (101 MHz, DMSO-d6) δ (ppm): 158.0, 147.2, 143.2, 134.9, 131.8, 131.2, 130.4, 129.5, 128.8, 127.6, 122.3, 121.86, 120.8, 119.4, 116.8, 56.6, 55.6, 54.5. HR-MS (positive mode, m / z) for [QYC] + , calculated value: 424.1867, found: 424.1846.

[0106] Fluorescence performance detection of copper ion fluorescent probe

[0107] I. UV-visible spectral response to Cu 2+ Using C2H5OH / HEPES buffer solution as the solvent, a probe ZYC solution with a concentration of 10 μmol / L was prepared. The C2H5OH / HEPES buffer solution was prepared from ethanol and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in a volume ratio of 25:75, and its pH value was 7.4.

[0108] In this invention, the UV-visible spectral titration method was used to study the absorption spectral response of probe ZYC to Cu

[0109] As shown in the main figure of (A), in the UV absorption spectra of the probe ZYC solution (10 μmol / L) at different concentrations of Cu 2+ (0 - 20 μmol / L), at a wavelength of 520 nm, the absorbance of the probe ZYC solution was the highest. Based on this, the absorbance of the probe ZYC solution (10 μmol / L) at 520 nm was studied as a function of the concentration of Cu Figure 10 (0 - 20 μmol / L) (upper right inset). It can be seen that as the concentration of Cu 2+ increased, the absorbance of the probe ZYC solution gradually decreased. When the concentration of Cu 2+ reached 20 μmol / L, the absorbance of the probe ZYC solution decreased significantly. 2+ concentration increased, the absorbance of the probe ZYC solution gradually decreased. When Cu 2+When the concentration reaches 10 μmol / L, the absorbance of the probe ZYC solution hardly changes. At the same time, as the concentration of Cu 2+ increases, the color of the probe ZYC solution changes from pink to light orange-red (upper left inset).

[0110] The present invention also studied the response of the probe ZYC (10 μmol / L) to various common metal ions (including Pb 2+ , Ag + , Cs + , Cu 2 + , Al 3+ , Ba 2+ , Fe 3+ , Hg 2+ , Ca 2+ , Cd 2+ , K + , Li + , Ca 2+ , Cd 2+ , Na + , Ni 2+ , Mn 2+ , Mg 2+ and Zn 2+ , with the concentration of each metal ion being 20 μmol / L). As shown in Figure 10 (B), only when Cu 2+ (20 μmol / L) is added, the absorption peak of the probe ZYC solution changes significantly, while the addition of other metal ions has little effect on the absorption peak of the probe ZYC solution, indicating that the probe ZYC can specifically respond to Cu 2+ .

[0111] II. Fluorescence spectral response to Cu 2+

[0112] Using C2H5OH / HEPES buffer solution as the solvent, the probe ZYC with a concentration of 10 μmol / L was prepared. The C2H5OH / HEPES buffer solution was prepared from ethanol and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in a volume ratio of 25:75, and its pH value was 7.4.

[0113] The present invention used fluorescence spectral titration to study the response of the probe ZYC to Cu 2+ . As shown in Figure 11 (A), for different concentrations of Cu 2+ ​In the fluorescence spectrum of the probe ZYC solution (10 μmol / L) at (0 - 20 μmol / L), the fluorescence intensity of the probe ZYC solution is the highest at a wavelength of 520 nm. Based on this, the change in the fluorescence intensity of the probe ZYC solution (10 μmol / L) at 520 nm with the concentration of Cu 2+ (0 - 20 μmol / L) was studied (upper right inset). It can be seen that as the concentration of Cu 2+ increases, the fluorescence intensity gradually decreases. When the concentration of Cu 2+ reaches 10 μmol / L, the fluorescence intensity of the probe ZYC solution hardly changes anymore. At the same time, as the concentration of Cu 2+ increases, the fluorescence of the probe ZYC solution changes from red to non-fluorescent (upper left inset).

[0114] This invention studied the fluorescence spectral response of the probe ZYC to various common metal ions (including Pb 2+ , Ag + , Cs + , Cu 2+ , Al 3+ , Ba 2+ , Fe 3 + , Hg 2+ , Ca 2+ , Cd 2+ , K + , Li + , Ca 2+ , Cd 2+ , Na + , Ni 2+ , Mn 2+ , Mg 2+ and Zn 2+ , and the concentration of each metal ion was 20 μmol / L). As shown in Figure 11 (B), only when Cu 2+ (20 μmol / L) was added, the fluorescence intensity of the probe ZYC solution (10 μmol / L) was significantly quenched, while the influence of other metal ions on the fluorescence intensity of the probe ZYC solution was not significant, indicating that the probe ZYC can specifically respond to Cu 2+ .

[0115] This invention studied the fluorescence spectral response of the probe ZYC in the presence of different metal ions (including Pb 2+ , Ag + , Cs + , Cu 2+ , Al 3+ , Ba 2+ , Fe 3+ , Hg 2+ , Ca 2+ , Cd2+ , K + , Li + , Ca 2+ , Cd 2+ , Na + , Ni 2+ , Mn 2+ , Mg 2+ and Zn 2+ ), the fluorescence response of probe ZYC to Cu 2+ is shown in Figure 11 (C). In the presence of other metal ions (20 μmol / L), the probe ZYC solution (10 μmol / L) has little effect on the recognition performance of Cu 2+ . The results show that probe ZYC can still specifically detect Cu 2+ .

[0116] The present invention studies the kinetic stability of probe ZYC, as shown in Figure 11 (D). In the C2H5OH / HEPES buffer solution, the fluorescence intensity of the probe ZYC solution (10 μmol / L) (λ ex = 520 nm, λ em = 660 nm) remains unchanged within 30 hours, indicating that probe ZYC has high kinetic stability.

[0117] Furthermore, the present invention studies the influence of different pH conditions on the fluorescence intensity of probe ZYC and the recognition of Cu 2+ by probe ZYC. As shown in Figure 12 (A), the probe ZYC solution (10 μmol / L) maintains stable and strong fluorescence emission in the range of pH 3 - 10 and shows good recognition ability for Cu 2+ (20 μmol / L) in the range of pH 5 - 9.5.

[0118] The present invention studies the response time of probe ZYC to Cu 2+ . As shown in Figure 12 (B), when Cu 2+ (3, 5, 7, 10, 15 μmol / L) is added to the probe ZYC solution (10 μmol / L), the fluorescence intensity of the probe ZYC solution rapidly decreases and reaches equilibrium within 30 s, indicating that probe ZYC can quickly detect Cu 2+ , meeting the requirements of practical applications.

[0119] The present invention also studies the relationship between the fluorescence intensity of probe ZYC and the concentration of Cu 2+ , as shown in Figure 12As shown in (C), the fluorescence intensity of the probe ZYC solution (2 μmol / L) at 660 nm has a good linear relationship with the concentration of Cu 2+ (0 - 0.55 μmol / L) (R 2 = 0.9872). The detection limit (LOD) was calculated to be 11.7 nmol / L according to LOD = 3σ / K, indicating that the sensitivity of the probe ZYC can meet the detection requirements for Cu in real water samples and food samples. 2+

[0120] III. Sensing mechanism for detecting Cu 2+

[0121] In order to study the sensing mechanism of the probe ZYC for detecting Cu 2+ , its sensing mechanism was verified by high-resolution mass spectrometry HR-MS. As shown in Figure 13 (A), after adding Cu to the probe ZYC solution until the reaction was complete (the color changed from pink to light orange-red), the molecular ion peak at m / z = 466.1708 disappeared, and a new molecular ion peak appeared at m / z = 527.0950 and corresponded to the complex product of ZYC-Cu 2+ (ZYC-Cu 2+ , m / z, calculated: 527.0861); 2+

[0122] In addition, its sensing mechanism was further verified by in-situ 1 1H NMR titration. After adding CuSO4 to the D2O / DMSO-d6 mixed solution (a solution composed of heavy water and deuterated dimethyl sulfoxide) until the reaction was complete (the color changed from pink to light orange-red), a 1H NMR titration experiment was carried out. As shown in 1 (B), it was found that the characteristic proton signal H1 of the phenolic hydroxyl -OH disappeared, while the characteristic proton signals H2 of the two alcoholic hydroxyl -OH did not disappear. This titration analysis indicates the coordination mechanism of the probe ZYC for Cu Figure 13 . 2+

[0123] Moreover, the sensing mechanism of the probe ZYC for detecting Cu 2+ was also studied by the Job's curve, complexation constant standard curve, and quenching constant standard curve after the probe ZYC recognized Cu 2+ . As shown in Figure 14 (A), the coordination relationship between the probe ZYC and Cu 2+ is 1:1. As shown in Figure 14 (B) and Figure 14 (C), the results of the complexation constant standard curve and quenching constant curve also indicate that the probe ZYC recognizes Cu 2+It is the quenching mechanism of "ON - OFF".

[0124] Example 4

[0125] This example provides a kit, which consists of a copper ion fluorescent probe, reaction medium paper, a solvent, and an ultraviolet detection lamp.

[0126] (1) Dissolve the copper ion fluorescent probe ZYC in methanol to obtain a 0.1 mmol / L probe solution. Soak the filter paper in the probe solution for 5 minutes, take it out and let it dry naturally to obtain a copper ion test paper.

[0127] (2) Soak the copper ion test paper in copper ion solutions with concentrations of 0, 15 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 70 μmol / L, and 80 μmol / L respectively, and obtain a fluorescence color comparison card according to the color change of the copper ion test paper under 365 nm ultraviolet light.

[0128] Comparative Example 1

[0129] This comparative example is basically the same as Example 4, and the only difference is that: in step (3), a color comparison card is obtained according to the color change of the copper ion test paper under natural light.

[0130] As Figure 16 shown, use the color recognition software of a smartphone to extract the R / (G + B) value of the test paper from Figure 16 (A) The color comparison card of Comparative Example 1 (upper figure) and the fluorescence color comparison card of Example 4 (lower figure) and obtain the Cu 2+ concentration calibration curve. It can be seen from the figure that the accuracy of the fluorescence color comparison card (D) is better than that of the color comparison card (C). Therefore, a kit is selected consisting of a fluorescence color comparison card and an ultraviolet detection lamp.

[0131] Wipe the surface of senna leaves, zongzi leaves, leeks, and garlic sprouts after spraying deionized water with the copper ion test paper of Example 4, and then compare the copper ion test paper with the fluorescence color comparison card. As Figure 17 (A)-(D) shown, the copper ion concentrations in the detected senna leaves, zongzi leaves, leeks, and garlic sprouts all exceed the standard.

[0132] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A copper ion fluorescent probe, characterized in that: The structural formula of the copper ion fluorescent probe is: Wherein, R is an electron-withdrawing group containing an active methyl group.

2. The copper ion fluorescent probe according to claim 1, characterized in that The R is selected from the following groups:

3. A method for preparing the copper ion fluorescent probe according to claim 1 or 2, characterized in that: The following steps are involved: In a first organic solvent, 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and compound R are mixed, and a catalyst is added to condense and reflux to perform a Knoevenagel condensation reaction to obtain a copper ion fluorescent probe; The compound R is an organic compound containing an active methyl group.

4. The method for preparing the copper ion fluorescent probe according to claim 3, characterized in that: The molar ratio of the 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde to the compound R is 1:1-1.2; The ratio of the sum of the molar amounts of the 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and the compound R to the molar amount of the catalyst is 1:n, wherein n≥3; The first organic solvent is ethanol; The catalyst is selected from one of piperidine and pyridine; The condensation reflux temperature is 80-90° C. and the time is 12-14 hours.

5. The method for preparing the copper ion fluorescent probe according to any one of claims 3 to 4, characterized in that: The compound R is selected from the following structures:

6. A copper ion detection test paper, characterized in that, The invention comprises a reaction medium paper and a copper ion fluorescent probe loaded on the reaction medium paper, wherein the copper ion fluorescent probe is the copper ion fluorescent probe according to claim 1 or 2 or the copper ion fluorescent probe prepared by the method according to any one of claims 3 to 5.

7. A method for preparing the copper ion detection test paper according to claim 6, characterized in that: The following steps are involved: The reaction medium paper is immersed in the copper ion fluorescent probe solution and then dried to obtain the copper ion detection test paper.

8. The method for preparing the copper ion detection test paper according to claim 7, characterized in that, The copper ion fluorescent probe solution is obtained by dissolving the copper ion fluorescent probe in a second organic solvent; The concentration of the copper ion fluorescent probe solution is 0.1-0.3 mmol / L; The second organic solvent includes one or more of methanol, ethanol, acetone, and tetrahydrofuran; The soaking time is 5-10 minutes.

9. A kit, characterized in that: It comprises a copper ion fluorescent probe, a reaction medium paper, a solvent and an ultraviolet detection lamp, wherein the copper ion fluorescent probe is the copper ion fluorescent probe according to claim 1 or 2 or the copper ion fluorescent probe prepared by the method according to any one of claims 3 to 5, and the solvent is used to dissolve the copper ion fluorescent probe; Or comprising copper ion detection test paper, a fluorescent colorimetric card and an ultraviolet detection lamp, wherein the copper ion detection test paper is the copper ion detection test paper according to claim 6 or the copper ion detection test paper prepared by the method according to any one of claims 7-8.

10. A use of the kit according to claim 9, characterized in that: The kit is used for detecting copper ions in food materials, medicinal materials or water environments.

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