A copper ion fluorescent probe, a copper ion test strip, their preparation method and reagent kit, and the application of the reagent kit.

Abstract

This invention relates to the field of copper ion detection technology, and discloses a copper ion fluorescent probe, a copper ion test strip, a preparation method and kit thereof, and the application of the kit. The structural formula of the copper ion fluorescent probe is: where R is an electron-withdrawing group containing an active methyl group. The kit prepared based on the copper ion fluorescent probe of this invention is portable and low-cost, and can perform qualitative or semi-quantitative real-time rapid detection of copper ions without the need for professional personnel or specialized equipment, with the assistance of an ultraviolet detection lamp.

Description

Technical Field

[0001] This invention relates to the field of copper ion detection technology, and in particular to a copper ion fluorescent probe, a copper ion test strip, a method for preparing the same, a reagent kit, and the application of the reagent kit. Background Technology

[0002] Copper ions (Cu) 2+ Copper ions are a widely distributed heavy metal element in nature. To avoid heavy metal poisoning, the use of copper ions in food is strictly limited. However, some businesses still use copper sulfate, taking advantage of its coloring properties in traditional Chinese medicine and its bactericidal and preservative effects on vegetables. This easily leads to seriously excessive levels of copper ions in traditional Chinese medicine products and vegetables, causing serious health, safety, and environmental problems. Furthermore, excessive accumulation of copper ions in water also poses a significant threat to the surrounding environment.

[0003] Copper ions pose a significant threat to food and environmental safety. To increase the detection of copper ions in food and the environment, rapid detection and analysis of copper ions are needed. However, current copper ion detection methods require specialized personnel and equipment, resulting in complex procedures, high costs, and limitations in personnel and available space. Therefore, finding a low-cost, rapid on-site detection method for copper ions 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 test strip, a preparation method thereof, a reagent kit, and the application of the reagent kit, to solve the problem that the existing copper ion detection process is complex, costly, and requires a high level of expertise, thus preventing rapid on-site detection.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a copper ion fluorescent probe, the structural formula of which 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] Secondly, the present invention provides a method for preparing the copper ion fluorescent probe according to any one of the above claims, comprising the following steps:

[0012] In a first organic solvent, 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and compound R were mixed, and a catalyst was added and refluxed to carry out a 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 compound R is 1:1-1.2.

[0015] Preferably, the molar 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 condensation reflux temperature is 80-90℃ and the time is 12-14h.

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

[0020]

[0021] Thirdly, 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 copper ion fluorescent probe described above or a copper ion fluorescent probe prepared by any of the methods described above.

[0022] Fourthly, the present invention provides a method for preparing copper ion detection test strips, comprising the following steps: immersing reaction medium paper in the copper ion fluorescent probe solution and then drying it to obtain copper ion detection test strips.

[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 minutes.

[0027] Fifthly, the present invention provides a kit comprising a copper ion fluorescent probe, reaction medium paper, solvent, and ultraviolet detection lamp, wherein the copper ion fluorescent probe is the copper ion fluorescent probe described above or a copper ion fluorescent probe prepared by any of the methods described above, and the solvent is used to dissolve the copper ion fluorescent probe.

[0028] Alternatively, the kit may include copper ion test strips, a fluorescent colorimetric card, and a UV detection lamp, wherein the copper ion test strips are the copper ion test strips described above or copper ion test strips prepared by any of the methods described above.

[0029] Sixthly, the present invention provides an application of the above-mentioned reagent kit, which is used for the detection of copper ions in food, medicinal materials or aquatic environments.

[0030] This invention provides a copper ion fluorescent probe, a copper ion test strip, a preparation method thereof, a reagent kit, and applications of the reagent kit. Compared with existing technologies, its advantages are as follows:

[0031] The reagent kit prepared based on the copper ion fluorescent probe of the present invention is portable and low in cost. It can perform qualitative or semi-quantitative real-time and rapid detection of copper ions without the need for professional personnel or specialized equipment, with the assistance of an ultraviolet detection lamp. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

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

[0034] Figure 2 The copper ion fluorescent probe ZYC of Example 1 of this invention 1 HNMR spectrum;

[0035] Figure 3 The copper ion fluorescent probe ZYC of Example 1 of this invention 13 C NMR spectrum;

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

[0037] Figure 5The copper ion fluorescent probe VIC of Example 2 of this invention 1 HNMR spectrum;

[0038] Figure 6 The copper ion fluorescent probe VIC of Example 2 of this invention 13 CNMR plot;

[0039] Figure 7 This is the high-resolution mass spectrum of the copper ion fluorescent probe QYC of Example 3 of the present invention;

[0040] Figure 8 The copper ion fluorescent probe QYC of Example 3 of this invention 1 HNMR spectrum;

[0041] Figure 9 The copper ion fluorescent probe QYC of Example 3 of this invention 13 CNMR plot;

[0042] Figure 10 The UV-Vis absorption spectrum of the copper ion fluorescent probe ZYC is shown in Figure (A), and the UV-Vis absorption spectrum of the copper ion fluorescent probe ZYC in the presence of multiple metal ions is shown in Figure (B).

[0043] Figure 11 The fluorescence spectrum of copper ion fluorescent probe ZYC is shown in Figure (A). The fluorescence spectrum of copper ion fluorescent probe ZYC in the presence of multiple metal ions is shown in Figure (B). The fluorescence response of copper ion fluorescent probe ZYC to copper ions in the presence of different metal ions is shown in Figure (C). The fluorescence stability curve of copper ion fluorescent probe ZYC is shown in Figure (D).

[0044] Figure 12 The diagram shows the effect of pH on the fluorescence response of the copper ion fluorescent probe ZYC (A), the response time of the copper ion fluorescent probe ZYC to different concentrations of copper ions (B), and the effect of copper ion concentration on the fluorescence intensity of the copper ion fluorescent probe ZYC (C).

[0045] Figure 13 High-resolution mass spectrum (A) of the copper ion fluorescent probe ZYC in the presence of copper ions. 1 Figure (B) shows the results of the 1H NMR titration experiment.

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

[0047] Figure 15 This is a schematic diagram of the reagent kit;

[0048] Figure 16 A schematic diagram of the colorimetric card and the fluorescent colorimetric card (A), a schematic diagram of the color of the colorimetric card and the fluorescent colorimetric card extracted using a smartphone (B), a calibration curve of the colorimetric card for copper ion concentration (C), a calibration curve of the fluorescent colorimetric card for copper ion concentration (D), and a schematic diagram of the process of detecting copper ion concentration using a kit (E).

[0049] Figure 17 A schematic diagram illustrating the detection of excessive copper ions in senna leaves (A), zongzi leaves (B), leeks (C), and garlic scapes (D) using copper ion test strips. Detailed Implementation

[0050] The present invention will be described below through specific embodiments. Those skilled in the art will understand that the specific embodiments below are merely illustrative and do not limit the scope of the invention in any way. Furthermore, in the following embodiments, unless otherwise specified, the reagents and equipment used are commercially available. If specific processing conditions and methods are not explicitly described in the later embodiments, conditions and methods known in the art can be used for processing.

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

[0052]

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

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

[0055]

[0056] In a second aspect, the present invention provides a method for preparing a copper ion fluorescent probe as described in any one of the above claims, comprising the following steps:

[0057] In a first organic solvent, 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde and compound R were mixed, and a catalyst was added and refluxed to carry out a Knauvengel condensation reaction to obtain a 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 and 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 includes the following steps: dissolving 3-chloromethyl-5-nitrosalicylic acid and diethanolamine in tetrahydrofuran, adding triethylamine to react, then refluxing, and after the reaction is completed, cooling to room temperature, filtering to obtain an orange-red solid, which is 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde.

[0061] The molar ratio of 3-chloromethyl-5-nitrosalicylic acid to diethanolamine is 1:1-1.2, for example, 1:1, 1:1.1, 1:1.2, etc.; the molar ratio of 3-chloromethyl-5-nitrosalicylic acid to triethylamine is 1:3-4, for example, 1:3, 1:3.5, 1:4, etc.; the reaction temperature is 0℃, and the reaction time is 50-70 min, for example, 50 min, 60 min, 70 min, etc.; the reflux temperature can be higher than the solvent temperature, and the reflux time is 15-17 h, for example, 15 h, 16 h, 17 h, etc.; filtration can be vacuum filtration, which is not particularly limited, as long as a solid product can be obtained.

[0062] In some embodiments of the present invention, the first organic solvent is ethanol, and there is no special limitation on the amount of the first organic solvent added, 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 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 condensation reflux temperature is 80-90°C, for example, 80°C, 85°C, 90°C, etc., and the condensation reflux time is 12-14h, for example, 12h, 13h, 14h, etc.

[0065] In some embodiments of the present invention, the preparation method of the copper ion fluorescent probe further includes the following steps: after the reaction is completed, the sample is cooled to room temperature, filtered, and dried to obtain a solid copper ion fluorescent probe.

[0066] In some embodiments of the present invention, the drying time is 24-30 hours, for example, 24 hours, 26 hours, 28 hours, 30 hours, etc. The drying method is not specifically limited, for example, it can be air drying at room temperature or drying in a general drying oven; the filtration method can be vacuum filtration, and the filtration method is not specifically limited and 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 a third aspect, the present invention provides a copper ion detection test strip, which 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 copper ion fluorescent probe described above or a copper ion fluorescent probe prepared by any of the methods described above.

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

[0071] In a fourth aspect, the present invention provides a method for preparing a copper ion detection test strip, comprising the following steps: immersing a reaction medium paper in the copper ion fluorescent probe solution and then drying it 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 a 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, which may 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 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc.

[0074] In a fifth aspect, the present invention provides a reagent kit.

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

[0076] In other embodiments of the present invention, the kit includes copper ion test strips, a fluorescent colorimetric card, and an ultraviolet detection lamp, wherein the copper ion test strips are the copper ion test strips described above or copper ion test strips prepared by any of the methods described above.

[0077] It should be noted that the main difference between the two kits is whether or not they include a fluorescent colorimetric card. Compared to the kit that includes a fluorescent colorimetric card, the kit that does not include a fluorescent colorimetric card allows for the preparation of the fluorescent colorimetric card and / or copper ion concentration standard curve card according to the range of copper ion concentration to be detected, offering greater flexibility. Furthermore, the kit can achieve more accurate copper ion concentration detection based on the copper ion concentration standard curve card.

[0078] In solutions that do not include a fluorescent colorimetric card, a fluorescent colorimetric card can be prepared by the user using the kit according to the following steps. The preparation method for the fluorescent colorimetric card is as follows: First, take a copper ion fluorescent probe, reaction medium paper, and solvent, and prepare copper ion test strips using the method described above; then, immerse multiple copper ion test strips in copper ion solutions of varying concentrations, and obtain a fluorescent colorimetric card based on the color of the copper ion test strips under ultraviolet light.

[0079] In some embodiments of the present invention, copper ion test strips are immersed in a series of copper ion solutions of different concentrations. The immersion time is 5-10 minutes, for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, etc. The concentration of the series of copper ion solutions can be adjusted according to the actual situation and is not specifically limited. 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 color of each copper ion test strip is obtained under ultraviolet light, and a fluorescent colorimetric card is prepared.

[0080] In some embodiments of the present invention, the R / (G+B) value of the test paper is extracted from the fluorescent colorimetric card using the color recognition software of a smartphone to obtain the copper ion concentration calibration curve, which can be prepared into a copper ion concentration standard curve card.

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

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

[0083] In a fifth aspect, the present invention provides an application of the above-described reagent kit for the detection of copper ions in food, medicinal materials, or aquatic environments.

[0084] Understandably, when testing for copper ions in food or medicinal materials, sampling is completed by spraying water onto the surface of the food or medicinal materials and then wiping the surface with copper ion test paper. When testing for copper ions in water samples, sampling is completed by dripping a well-mixed water sample onto copper ion test paper and letting it stand. The standing time is generally less than 1 minute, and there are no special restrictions on this.

[0085] like Figure 16 As shown in (E), if a kit is needed for qualitative or semi-quantitative detection of copper ion concentration, the sampled copper ion test strip can be compared with the fluorescent colorimetric card under 365nm ultraviolet light irradiation. If a kit is needed for more accurate quantitative detection of copper ion concentration, the R / (G+B) value of the sampled copper ion test strip needs to be extracted using an ultraviolet dark box and a smartphone, and then the value can be read using the copper ion concentration standard curve card.

[0086] The technical solutions of this invention will be clearly and completely described below with reference to specific embodiments. The embodiments of this application are only examples, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0087] Example 1

[0088] This embodiment provides a method for preparing the copper ion fluorescent probe ZYC, and the specific steps are as follows:

[0089] (1) 3-chloromethyl-5-nitrosalicylic aldehyde (1.0 g, 4.64 mmol) and diethanolamine (0.49 g, 4.66 mmol) were dissolved in 50 mL of tetrahydrofuran, and 2 mL of triethylamine (1.46 g, 14.39 mmol) was added. The mixture was reacted at 0 °C for 60 min, and then refluxed for 17 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The filtrate was evaporated to dryness to obtain 1.27 g of orange-red solid, namely 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrosobenzaldehyde, with a yield of 96%.

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

[0091]

[0092] (2) 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) were dissolved in 15 mL of anhydrous ethanol, and then 0.2 mL of pyridine was added and refluxed for 12 h. After cooling to room temperature, the mixture was filtered and dried 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 for the copper ion fluorescent probe ZYC is as follows:

[0094]

[0095] like Figure 1-3 The images shown are the high-resolution mass spectra of the copper ion fluorescent probe ZYC. 1 HNMR spectrum and 13 The C NMR spectrum shows that... 1 H NMR (400MHz, DMSO-d6) δ (ppm): 9.53 (s, 1H), 8.61 (d, J=3.1Hz, 1H), 8.37-8.04 (m, 2H), 7.90 (d, J= 15.8Hz,1H),5.26(s,2H),4.35(s,2H),3.78(t,J=5.0Hz,4H),3.22(t,J=4.9Hz,4H),1.80(s,6H). 13 C NMR(101MHz,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 embodiment provides a method for preparing a copper ion fluorescent probe VIC. The preparation method of the copper ion fluorescent probe VIC is basically the same as that in Example 1, except that the compound R is different.

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

[0099]

[0100] like Figure 4-6 The images shown are the high-resolution mass spectra of the copper ion fluorescent probe VIC. 1 HNMR spectrum and 13 The C NMR spectrum shows that... 1 H NMR(400MHz,DM SO-d6)δ(ppm):8.39(d,J=3.0Hz,1H),7.98(d,J=3.0Hz,1H),7.69(d,J=16.1Hz,1H),7.47(d,J=16.1Hz,1H),6.79( s,1H),5.20(s,2H),4.30(s,2H),3.74(t,J=5.3Hz,4H),3.12(t,J=5.3Hz,4H),2.56(d,J=28.7Hz,4H),1.02(s,6H). 13 C NMR(101MHz,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(posi tive mode,m / z)for[VIC-H + ] - Calculated value: 451.1987, found: 451.3005.

[0101] Example 3

[0102] This embodiment provides a method for preparing a copper ion fluorescent probe QYC. The preparation method of the copper ion fluorescent probe QYC is basically the same as that in Example 1, except that the compound R is different.

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

[0104]

[0105] like Figure 7-9 The images shown are the high-resolution mass spectra of the copper ion fluorescent probe QYC. 1 HNMR spectrum and 13 The C NMR spectrum shows that... 1H NMR (400MHz, DMSO-d6) δ (ppm): 10.04 (s, 1H), 8.90 (d, J = 9.1Hz, 1H), 8.67 (d, J = 15.2Hz, 1H), 8.56-8.47 (m, 2H), 8.44 (d, J = 9.0Hz, 1H), 8 .32-8.21(m,2H),8.17-8.07(m,2H),7.88(t,J=7.5Hz,1H),5.28(s,2H),4.42(s,3H),4.40(s,2H),3.80(s,4H),3.25(t,J=5.0Hz,4H). 13 C NMR(101MHz,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 probes

[0107] I. For Cu 2+ UV-Vis spectral response

[0108] A probe ZYC solution with a concentration of 10 μmol / L was prepared using C2H5OH / HEPES buffer solution as the solvent. The C2H5OH / HEPES buffer solution was prepared by mixing ethanol and (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) in a volume ratio of 25:75, and its pH value was 7.4.

[0109] This invention employs ultraviolet-visible spectroscopy titration to study the effect of probe ZYC on Cu. 2+ The absorption spectrum response, such as Figure 10 As shown in the main graph of (A), different concentrations of Cu 2+ In the UV absorption spectrum of the probe ZYC solution (10 μmol / L) at (0-20 μmol / L), the absorbance of the probe ZYC solution is highest at a wavelength of 520 nm. Based on this, the absorbance of the probe ZYC solution (10 μmol / L) at 520 nm as a function of Cu was investigated. 2+ The change in concentration (0-20 μmol / L) (top right inset) shows that as Cu... 2+ As the concentration increases, the absorbance of the probe ZYC solution gradually decreases. 2+When the concentration of Cu reaches 10 μmol / L, the absorbance of the probe ZYC solution hardly changes, while with the increase of Cu... 2+ As the concentration increased, the color of the probe ZYC solution changed from pink to light orange-red (top left inset).

[0110] This invention also investigated the effects of the probe ZYC (10 μmol / L) on 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+ The response of various metal ions (at a concentration of 20 μmol / L) is as follows: Figure 10 As shown in (B), only when Cu is added... 2+ The absorption peak of the probe ZYC solution only showed a significant change at a concentration of 20 μmol / L, while the addition of other metal ions had little effect on the absorption peak of the probe ZYC solution, indicating that the probe ZYC can specifically respond to Cu. 2+ .

[0111] II. For Cu 2+ fluorescence spectral response

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

[0113] This invention uses fluorescence spectrometry titration to study the effect of probe ZYC on Cu. 2+ The response, such as Figure 11 As shown in (A), 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 was highest at a wavelength of 520 nm. Based on this, the fluorescence intensity of the probe ZYC solution (10 μmol / L) at 520 nm as a function of Cu was investigated. 2+ The change in concentration (0-20 μmol / L) (top right inset) shows that as Cu... 2+ As the concentration increases, the fluorescence intensity gradually decreases. 2+ When the concentration reaches 10 μmol / L, the fluorescence intensity of the probe ZYC solution hardly changes, while with Cu... 2+ As the concentration increased, the fluorescence of the probe ZYC solution changed from red to non-fluorescent (top left inset).

[0114] This invention investigates the effects of the probe ZYC on 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+ The fluorescence spectral response of various metal ions (at a concentration of 20 μmol / L) is as follows: Figure 11 As shown in (B), only when Cu is added... 2+ At a concentration of 20 μmol / L, the fluorescence intensity of the probe ZYC solution (10 μmol / L) only showed significant quenching, while other metal ions had little effect on the fluorescence intensity of the probe ZYC solution, indicating that the probe ZYC can specifically respond to Cu. 2+ .

[0115] This invention studies the effects 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+ In the presence of ) probe ZYC to Cu 2+ fluorescence response, such as Figure 11 As shown in (C), in the presence of other metal ions (20 μmol / L), the probe ZYC solution (10 μmol / L) on Cu 2+ The recognition performance was not significantly affected, indicating that the probe ZYC can still specifically detect Cu even in the presence of other potentially interfering metal ions. 2+ .

[0116] This invention investigates the kinetic stability of the probe ZYC, such as... Figure 11 As shown in (D), the fluorescence intensity (λ) of the probe ZYC solution (10 μmol / L) in C2H5OH / HEPES buffer solution. ex =520nm, λ em The wavelength (660 nm) remained unchanged over 30 hours, indicating that the probe ZYC has high kinetic stability.

[0117] Furthermore, this invention investigated the effect of different pH conditions on the fluorescence intensity of probe ZYC and the effect of probe ZYC on Cu. 2+ The impact of recognition, such as Figure 12 As shown in (A), the probe ZYC solution (10 μmol / L) maintained stable and strong fluorescence emission in the pH range of 3-10, and exhibited good fluorescence emission against Cu in the pH range of 5-9.5. 2+ (20 μmol / L) showed good recognition ability.

[0118] This invention studies the effect of probe ZYC on Cu 2+ Response time, such as Figure 12 As shown in (B), when Cu is added to the probe ZYC solution (10 μmol / L) 2+ At concentrations of 3, 5, 7, 10, and 15 μmol / L, the fluorescence intensity of the ZYC probe solution decreased rapidly and reached equilibrium within 30 seconds, indicating that the ZYC probe can rapidly detect Cu. 2+ It meets the requirements of practical applications.

[0119] This invention also investigated the fluorescence intensity of the probe ZYC in relation to Cu. 2+ The relationship between concentrations, such as Figure 12As shown in (C), the fluorescence intensity of the probe ZYC solution (2 μmol / L) at 660 nm is similar to that of Cu. 2+ The concentration (0-0.55 μmol / L) showed a good linear relationship (R0). 2 =0.9872), and the detection limit (LOD) was calculated to be 11.7 nmol / L based on LOD = 3σ / K, indicating that the sensitivity of probe ZYC is sufficient for Cu in real water and food samples. 2+ Testing requirements.

[0120] III. Detection of Cu 2+ Sensing mechanism

[0121] This invention aims to study the detection of Cu by the ZYC probe. 2+ The sensing mechanism was verified by high-resolution mass spectrometry (HR-MS), such as... Figure 13 As shown in (A), Cu was added to the probe ZYC solution. 2+ After 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, which coincided with the ZYC-Cu peak. 2+ The complex product (ZYC-Cu) 2+ , m / z, calculation: 527.0861) corresponding;

[0122] In addition, through in-situ 1 ¹H NMR titration further verified its sensing mechanism. CuSO₄ was added to a D₂O / DMSO-d₆ mixed solution (a solution of heavy water and deuterated dimethyl sulfoxide) until the reaction was complete (the color changed from pink to light orange-red), and then... 1 HNMR titration experiments, such as Figure 13 As shown in (B), the characteristic proton signal H1 of the phenolic hydroxyl group -OH disappeared, while the characteristic proton signal H2 of the two alcoholic hydroxyl groups -OH did not disappear. This titration analysis indicates that the probe ZYC is effective against Cu. 2+ The coordination mechanism.

[0123] Furthermore, Cu was identified using the ZYC probe. 2+ The Job's curve, complexation constant standard curve, and quenching constant standard curve were used to study the detection of Cu by the probe ZYC. 2+ The sensing mechanism, such as Figure 14 As shown in (A), the probes ZYC and Cu 2+ A 1:1 coordination relationship, such as Figure 14 (B) and Figure 14 As shown in (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 "ON-OFF" quenching mechanism.

[0124] Example 4

[0125] This embodiment provides a kit consisting of a copper ion fluorescent probe, reaction medium paper, solvent, and ultraviolet detection lamp.

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

[0127] (2) The copper ion test paper was immersed 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 a fluorescent colorimetric card was obtained based on 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, except that in step (3), a colorimetric card is obtained based on the color change of the copper ion test paper under natural light.

[0130] like Figure 16 As shown, using smartphone color recognition software from Figure 16 (A) Extract the R / (G+B) value of the test strip from the colorimetric card of Comparative Example 1 (top image) and the fluorescent colorimetric card of Example 4 (bottom image) and obtain the Cu value. 2+ The concentration calibration curve shows that the accuracy of the fluorescence colorimetric card (D) is better than that of the colorimetric card (C). Therefore, the fluorescence colorimetric card and a UV detection lamp were selected to form the kit.

[0131] The copper ion test strip from Example 4 was wiped on the surfaces of senna leaves, zongzi leaves, chives, and garlic scapes after being sprayed with deionized water. The copper ion test strip was then compared with a fluorescent colorimetric card. Figure 17 As shown in (A)-(D), the concentrations of copper ions in the tested senna leaves, zongzi leaves, chives, and garlic scapes all exceeded the standard.

[0132] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify 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:

2. A method for preparing the copper ion fluorescent probe according to claim 1, 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 were mixed, and a catalyst was added and refluxed to carry out a Knauvengel condensation reaction to obtain a copper ion fluorescent probe. The compound R is 3. The method for preparing the copper ion fluorescent probe according to claim 2, characterized in that, The molar ratio of 3-((bis(2-hydroxyethyl)amino)methyl)-2-hydroxy-5-nitrobenzaldehyde to compound R is 1:1-1.2; The molar 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; The first organic solvent is ethanol; The catalyst is selected from piperidine and pyridine; The condensation reflux temperature is 80-90℃, and the time is 12-14h.

4. A copper ion detection test strip, characterized in that, It 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 copper ion fluorescent probe of claim 1 or the copper ion fluorescent probe prepared by the method of any one of claims 2-3.

5. A method for preparing the copper ion detection test strip according to claim 4, characterized in that, The following steps are involved: The reaction medium paper is soaked in the copper ion fluorescent probe solution and then dried to obtain copper ion detection paper.

6. The method for preparing the copper ion detection test strip according to claim 5, 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.

7. A reagent kit, characterized in that, The apparatus includes a copper ion fluorescent probe, reaction medium paper, solvent, and ultraviolet detection lamp, wherein the copper ion fluorescent probe is the copper ion fluorescent probe according to claim 1 or the copper ion fluorescent probe prepared by the method according to any one of claims 2-3, and the solvent is used to dissolve the copper ion fluorescent probe. It may include copper ion test strips, fluorescent colorimetric cards, and ultraviolet detection lamps, wherein the copper ion test strips are the copper ion test strips of claim 4 or the copper ion test strips prepared by the method of any one of claims 5-6.

8. The application of the reagent kit according to claim 7, characterized in that, The kit is used for the detection of copper ions in food, medicinal materials, or aquatic environments.

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