Isophorone derivative Y as well as preparation method and application thereof

Abstract

According to the present invention, the isophorone derivative fluorescent probe Y is synthesized, the excitation wavelength of the Y is 432 nm, the emission wavelength is 623 nm, the Stokes shift can achieve 191 nm, and the good light stability is provided; through fluorescence quenching response, Y can detect Cu < 2 + > with high selectivity; cu < 2 + > is added to Y in a DMF / H2O solution, fluorescence at 623 nm is remarkably quenched, and the detection limit is 1.78 * 10 <-7 > M; in addition, the Y-Cu < 2 + > complex is used as a combined probe, and S < 2-> can be selectively detected through a competitive substitution reaction; y and Y-Cu < 2 + > have been successfully applied to labeling recovery detection of Cu < 2 + > and S < 2-> in an actual water sample; the real-time detection of Cu < 2 + > and S < 2-> is realized by using a test strip and a smart phone as auxiliary means; in addition, Y can effectively detect content changes of exogenous and endogenous Cu < 2 + > and S < 2-> in A2780 cells.

Description

Technical Field

[0001] The present invention relates to the technical fields of organic synthesis and ion detection, and particularly relates to an isophorone derivative Y, a preparation method thereof and an application thereof. Background Art

[0002] Cu 2+ As the third most abundant metal ion in the human body, it plays a crucial role in a variety of biological processes, such as participating in the degradation of reactive oxygen species, catalyzing metal enzyme cofactors, and signal transduction. Cu in the body 2+ imbalance can lead to serious diseases, such as diarrhea, vomiting, Alzheimer's disease and other diseases. On the other hand, with the excessive use of Cu in daily life and industrial production, 2+ Cu 2+ will flow into lakes and oceans, causing a series of water environment problems. In addition, Cu 2+ is easily accumulated in aquatic organisms and causes serious food problems. It can also enter the human body through the food chain, posing a hazard to human health. Therefore, establishing a method for accurately quantifying Cu 2+ detection is of great significance for environmental protection and human health. Among many analytical detection methods, optical probes have received extensive attention due to their high sensitivity, small damage to analytes, convenient application in bioimaging and "naked eye" detection and other advantages.

[0003] Hydrogen sulfide is a toxic gas and an important environmental pollutant. Human exposure to low concentrations of H2S may cause certain irritation to the eyes and respiration. More seriously, high concentrations of H2S can be fatal in a short time. H2S exists in wastewater and is released into environmental water bodies, polluting the environment. In addition, H2S is naturally produced from foods containing organic sulfur, such as meat, eggs and vegetables. It is produced by the metabolites of sulfur-containing bacteria in meat and is considered to be one of the main volatiles during the meat spoilage process. H2S has recently been introduced as an indicator for determining food freshness. Therefore, developing a simple and efficient H2S detection method is of great significance. However, most H2S fluorescent probes exhibit relatively small Stokes shifts (<100 nm), and other biological thiols are prone to interfere with them. CuS is considered to be a very stable substance, and many copper complexes are used for the detection of S 2+ ions based on the substitution strategy of Cu 2- chelating ligands. Therefore, the use of the complex formed by a fluorescent molecule and Cu 2+ as a combined probe for detecting sulfide ions has attracted much attention. A number of studies have reported various types of fluorescent probes for detecting Cu 2+ and S 2- ions, and significant progress has been made in the structural design and application of continuous luminescence fluorescent probes. However, there are not many reports on near-infrared fluorescent probes. Summary of the Invention

[0004] The object of the present invention is to design and synthesize a more sensitive and highly selective fluorescent probe, namely an isophorone derivative Y, aiming to achieve efficient detection of Cu 2+ and S 2- and enhance practicality.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is: an isophorone derivative Y, whose structural formula is:

[0006]

[0007] The present invention also provides a preparation method of the above isophorone derivative Y. Weigh 5-[(3-dicyanomethylene-5,5-dimethyl)cyclohex-1-enyl]salicylaldehyde into a two-necked flask, dissolve it by reflux stirring with ethanol at 80 °C. After complete dissolution, add benzylamine, reflux and stir for 10 hours, cool to room temperature, store the obtained liquid in a refrigerator at 4 °C for two days. After the liquid precipitates solid, collect it by vacuum filtration, and wash the crude product three times with cold ethanol and methanol in turn to obtain isophorone derivative Y.

[0008] As a further limitation of the preparation method of the present invention, the molar ratio of 5-[(3-dicyanomethylene-5,5-dimethyl)cyclohex-1-enyl]salicylaldehyde to benzylamine is 1:1, and the molar volume ratio of benzylamine to ethanol is 3 mmol: 200 mL.

[0009] The present invention also provides the application of the above isophorone derivative Y in the detection of Cu 2+

[0010] As a further limitation of the above application, after drying the probe test strip loaded with 5 μM Y, immerse it into solutions containing different Cu 2+ concentrations respectively, and record the color change of the test strip under the irradiation of 365 nm ultraviolet light. As the Cu 2+ concentration increases, the color of the filter paper strip gradually changes from light red to purple under ultraviolet light.

[0011] As a further limitation of the above application, place the test sample containing Cu 2+ in a dark box to avoid light source interference, use a 365 nm 3W LED lamp as the excitation source, capture the fluorescence image with a smartphone camera at night, and use the application Color Desk program to process the image to obtain the corresponding RGB value. The RGB value shows a good linear relationship with the concentration of Y and Cu 2+

[0012] The present invention also provides the application of the above isophorone derivative Y in the detection of endogenous / exogenous Cu in cells​​2+ Applications in imaging, including the following steps: First, incubate cells with 20 μM of Y for 10 min, and significant strong fluorescence is observed in the red channel. Then, add different concentrations of Cu 2+ and process for 10 min. As the concentration of Cu 2+ increases, the fluorescence in the red channel gradually quenches.

[0013] The present invention also provides the application of the above-mentioned isophorone derivative Y-Cu 2+ complex in S 2- detection.

[0014] As a further limitation of the above application, immerse the dried test strip loaded with the Y-Cu 2+ complex into solutions with different S 2- concentrations. After drying, observe the color change under 365 nm ultraviolet light as follows: As the concentration of S 2- increases, the color of the filter paper strip changes from purple back to light red.

[0015] The present invention also provides the application of the above-mentioned isophorone derivative Y-Cu 2+ complex in intracellular / extracellular S 2- imaging. Incubate cells with 20 μM of Y and 22 μM of Cu 2+ for 10 min, add different concentrations of S 2- to the culture medium and incubate for 10 min. The fluorescence imaging change is as follows: As the concentration of S 2- gradually increases, the fluorescence intensity of the system at 623 nm increases significantly, showing a good linear relationship with S 2- in the concentration range of 1 - 15 μM, with R 2 = 0.9958. S 2- can be quantitatively detected by means of colorimetry.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention condenses isophorone derivatives with benzylamine to synthesize a novel near-infrared fluorescence probe Y, which can highly selectively detect Cu 2+ . Using Y-Cu 2+ as a combined probe, S 2- can be detected based on the principle of competitive substitution. Y has the advantages of high sensitivity, simple synthesis, good selectivity, large Stokes shift, etc. It can be used to detect Cu 2+ and S 2- in environmental solid water samples by using a fluorescence spectrometer, a portable test strip, and a smartphone, and Cu 2+ and S 2- in cells can be detected by fluorescence microscopy technology.Selective detection. Description of the Drawings

[0018] Figure 1 Spectral change diagram of Y (10 μM) after continuously adding Cu (0 - 14 μM). 2+ (0 - 14 μM) and then Y (10 μM).

[0019] Figure 2 Linear relationship diagram between the absorption ratio of Y (A / A) and [Cu]. 492nm / A 422nm ) and [Cu 2+ .

[0020] Figure 3 Fluorescence selectivity diagram of Y (5 μM) for various metals (50 μM).

[0021] Figure 4 Emission intensity diagram of Y (5 μM) at 623 nm in the presence of other related analytes (50 μM).

[0022] Figure 5 Fluorescence intensity diagrams of Y and Y - Cu at 623 nm at different pH values ([Y] = 5 μM, [Cu 2+ = 50 μM, [S 2+ = 50 μM). 2-

[0023] Figure 6 Fluorescence intensity change diagrams of Y, Y - Cu 2+ , Y - Cu 2+ - S 2- with time at 623 nm.

[0024] Figure 7 Emission spectral change diagram of Y (5 μM) after adding Cu (0 - 5.5 μM). 2+ (0 - 5.5 μM).

[0025] Figure 8 Relationship diagram between the emission intensity at 623 nm and the concentration of [Cu 2+ .

[0026] Figure 9 Effect diagram of adding Cu 2+ on the fluorescence lifetime of Y.

[0027] Figure 10 Fluorescence intensity change diagram of Y at 623 nm after alternately adding Cu 2+ and S 2- .

[0028] Figure 11 Fluorescence intensity diagrams of Y and Cu 2+Job's plot in DMF-HEPES buffer (1:9, v / v, 25 mM, pH 7.4) ([Y] + [Cu 2 + = 5 μM).

[0029] Figure 12 Benesi-Hilderbrand plot of the binding of Y to Cu 2+ .

[0030] Figure 13 Mass spectrum of Y-Cu 2+ in acetonitrile.

[0031] Figure 14 Proton nuclear magnetic resonance (1H NMR) spectrum of Y and Cu 2+ in d6-DMSO.

[0032] Figure 15 Infrared (IR) spectra of Y and the Y-Cu 2+ complex.

[0033] Figure 16 Possible binding modes and sensing mechanism diagram of Y and Cu 2+ .

[0034] Figure 17 Changes in the fluorescence emission spectrum of the system after adding S 2- ([Y] = 5 μM, [Cu 2+ = 5.5 μM).

[0035] Figure 18 Plot of the fluorescence emission intensity at 623 nm versus the concentration of [S 2- .[[]END]]

[0036] Figure 19 Changes in the ultraviolet absorption spectrum of the system after adding 12 μM S 2- .

[0037] Figure 20 Fluorescence intensity plot of Y-Cu 2+ in the presence of competitive anions and in the presence of S 2- .

[0038] Figure 21 Color change diagram of Cu 2+ ions on filter paper containing the probe Y under 365 nm ultraviolet light (upper row), and color change diagram of S 2- ions on filter paper containing the probe Y-Cu 2+ under 365 nm ultraviolet light (lower row).

[0039] Figure 22 Response of Y to Cu2+ Smartphone detection diagram.

[0040] Figure 23 Cell viability diagram after incubating A2780 cells for 24 h at different concentrations of Y (0 - 50 μM).

[0041] Figure 24 For the cellular imaging of Y on endogenous / exogenous Cu 2+

[0042] Figure 25 For Y - Cu 2+ on endogenous / exogenous S 2- Detailed implementation mode

[0043] The present invention will be further described below in conjunction with specific embodiments.

[0044] Example 1

[0045] Synthesis of isophorone derivative Y3 probe

[0046] Instruments and reagents

[0047] Table 1 Main experimental instruments

[0048]

[0049] Table 2 Main experimental reagents

[0050]

[0051] The synthesis route of Y is as follows:

[0052]

[0053] The synthesis methods of intermediates 1 - 3 are as follows:

[0054] Synthesis of compound 1: During the experiment, first take (2.1 mL, 13.8 mmol) of isophorone and (0.91 g, 13.8 mmol) of malononitrile and put them into a flask in turn. Then continue to add 30 mL of absolute ethanol to the flask and add an appropriate amount of piperidine. Stir the mixture in an oil bath at 65 °C for 10 hours. After the reaction is completed, cool it to room temperature, filter and dry to obtain a precipitated solid (1.2 g, yield 47%).

[0055] ​​Synthesis of Compound 2: Dissolve Compound 1 (2.0 g, 10.7 mmol) and 4-hydroxybenzaldehyde (1.6 g, 12.8 mmol) in 70 mL of anhydrous ethanol solution. Slowly add 5 drops of piperidine (0.25 mL) to the mixed liquid and reflux and stir for 4 hours. After the reaction is completed, pour the mixed solution into ice water at 4 °C to precipitate for 0.5 hour. Filter and dry to obtain orange solid Compound 2 (2.3 g, yield 70%).

[0056] Synthesis of Compound 3: Dissolve Compound 2 (0.58 g, 2 mmol) and hexamethylenetetramine (urotropine 0.84 g, 6 mmol) in 20 mL of trifluoroacetic acid, and heat in an oil bath at 90 °C for 10 hours. After the reaction is completed, purify by column chromatography using dichloromethane and methanol in a volume ratio of 200:1 as the eluent to obtain orange-red solid powder Compound 3 (yield 73%).

[0057] Synthesis of Y: Condense the synthesized Compound 3, namely 5-[(3-dicyanomethylene-5,5-dimethyl)cyclohexyl-1-ethenyl]salicylaldehyde, with benzylamine to obtain Y. Weigh Compound 3 (0.30 mmol, 95.44 mg) into a two-necked flask, dissolve it by refluxing and stirring with 20 mL of ethanol at 80 °C. After complete dissolution, add benzylamine (0.30 mmol, 32.12 μL), reflux and stir for 10 hours, cool to room temperature, store the resulting liquid in the refrigerator for two days. After the solid precipitates, collect it by vacuum filtration, and wash the crude product three times with cold ethanol and methanol in turn to obtain an orange-red solid compound (80 mg, yield 65.36%). 1 H NMR (600 MHz, DMSO) δ 12.87 (s, 1H), 8.96 (s, 1H), 8.81 (s, 1H), 8.24 (s, 3H), 8.16 (s, 2H), 7.92 (d, J = 9.9 Hz, 2H), 7.85 (d, J = 18.2 Hz, 1H), 7.52 (d, J = 20.2 Hz, 1H), 7.32 (s, 2H), 7.09 (d, J = 36.8 Hz, 1H), 6.87 (s, 1H), 2.62 (s, 2H), 2.56 (s, 2H), 1.03 (s, 6H). HRMS (ESI): C 27 H 25 N3O (M + H) + Theoretical value: 407.1998, actual value: 408.2070.

[0058] Example 2

[0059] Spectral determination method

[0060] The stock solution of Y was prepared in DMSO at a concentration of 1.0×10 -3M. Stock solutions (1.0×10 -2 M) of different metal salts were freshly prepared from the corresponding chloride or nitrate salts in doubly distilled water. Absorption and emission spectra were measured in DMF-HEPES buffer (3:2, v / v, 25 mM HEPES, pH 7.4). A certain volume of the Y stock solution was transferred into a 4-mL volumetric flask, and then an appropriate amount of the ion stock solution was added. The sample solution was diluted with DMF-HEPES buffer (1:9, v / v, 25 mM HEPES, pH 7.4) to reach the desired concentration. In the fluorescence spectroscopy test, the prepared solution to be detected was shaken well and allowed to stand for 10 minutes, and the absorption and emission spectra were recorded. The excitation wavelength was adjusted to 432 nm, and the slit widths of the excitation beam and the emission beam were set to 10 nm and 10 nm, respectively.

[0061] The recognition ability of compound Y for copper ions (Cu 2+ ) was investigated by UV-visible absorption spectroscopy. As Figure 1 shown, with the gradual addition of Cu 2+ , the absorption spectrum of Y showed significant changes. Y had a characteristic absorption peak at 422 nm. With the addition of Cu 2+ (0 - 14×10 -6 M), this peak gradually shifted to a longer wavelength of 492 nm. In addition, an isosbestic point appeared at 465 nm, indicating the formation of a stable complex between Y and Cu 2+ . Further, Figure 2 showed that in the range of Cu 2+ concentration from 2×10 -6 M to 14×10 -6 M, the absorbance ratio A 492nm / A 422nm showed a good linear correlation with the Cu 2+ concentration (R 2 = 0.9811), enabling the quantitative detection of Cu 2+ by colorimetry.

[0062] In the fluorescence spectroscopy test ( Figure 3 ), it was further observed that the addition of Cu 2+ significantly decreased the intensity of the probe Y at 623 nm. The introduction of other metal ions had little effect on the fluorescence spectrum. In addition, the metal ion competition method was used to study the effect of different metal ions on the recognition of Cu 2+ by Y. A 50 μM Cu 2+ solution was added to the Y solution, and then a 50 μM solution of different metal ions was added to the Y-Cu 2+ solution. The measurement results are as Figure 4As shown, after adding 50 μM of other metal ions, the fluorescence intensity of the Y-Cu 2+ system did not change significantly. These experiments indicate that, compared with other metal ions, the probe Y has a good selective recognition ability for Cu 2+ .

[0063] Fluorescence method for detecting Cu 2+

[0064] By measuring the emission intensities of Y, Y-Cu 2+ and Y-Cu 2+ -S 2- in solutions with different pH values, the influence of pH on the sensing performance of Y was investigated. As Figure 5 shown, the fluorescence intensity at 623 nm was not affected by pH. In the pH range of 3.0 - 9.0, the addition of Cu 2+ ions significantly decreased the emission intensity of Y. After adding S 2+ to Y-Cu 2- , the fluorescence recovered. Considering the pH of the biological system and the recognition ability of the probe, the pH of the test system was adjusted to 7.4. This enabled the detection of Cu 2+ by the probe Y and the detection of S 2+ by the combined probe Y-Cu 2- under physiological conditions.

[0065] Subsequently, the fluorescence titration method was used to investigate the detection sensitivity of Y for Cu 2+ . Figure 7 showed that the fluorescence intensity of Y at 623 nm decreased with increasing Cu 2+ concentration, and the red fluorescence of the solution was significantly quenched under ultraviolet light ( Figure 7 inset). In the range of 0.5 μM to 5.5 μM, the fluorescence intensity showed a good linear relationship with the Cu 2+ concentration ( Figure 8 ), and its linear correlation coefficient R 2 was 0.9940. Using the formula 3σ / s, the detection limit was calculated to be 1.77×10

[0066] M, which is more than 1000 times lower than the WHO-recommended Cu -8 drinking water standard (76 μM). Compared with other recently reported Cu 2+ fluorescent probes, Y can detect Cu 2+ with high sensitivity and specificity, and its detection limit is much lower. 2+ .

[0067] By measuring the fluorescence lifetime, it was found that the trend of Y did not change with the addition of Cu 2+ , proving that this fluorescence quenching belongs to static fluorescence quenching ( Figure 9)。Fluorescence titration experiment of Y with quencher Cu 2+ showed a good linear relationship in the range of (0.5 - 5 μM) ( Figure 8 ), conforming to the static enhancement of the Perrin model. According to the Perrin equation F0 / F = e -KsvQ The obtained fluorescence quenching efficiency value (Ksv) was 2.503×10 5 M -1 . In addition, we investigated the change of fluorescence intensity of the system at 623 nm over time ( Figure 6 ), and the results showed that Y had good photostability. Y responded very quickly to Cu 2+ and could achieve rapid detection of Cu 2+ .

[0068] By alternately adding Cu 2+ and S 2- to the probe solution, the reversibility of Y, Y + Cu 2+ for detecting Cu 2+ and S 2- was studied. It was observed that adding S 2+ to Y + Cu 2- caused a significant fluorescence enhancement due to the strong affinity of S 2- for Cu 2+ . Adding more Cu 2+ and S 2- to the same mixture, the fluorescence emission could be repeatedly turned on and off for more than 4 cycles ( Figure 10 ), indicating that Y was reversible for Cu 2+ .

[0069] Example 3

[0070] Study on the binding mode of Y and Cu 2+

[0071] Preparation of infrared samples

[0072] The preparation process of the infrared test samples was as follows: Accurately weigh 0.41 g (0.10 mmol) of Y and dissolve it in 10 mL of ethanol. After complete dissolution, slowly add 0.36 g (0.15 mmol) of Cu(NO3)2·3H2O and continuously stir the reaction at room temperature for 2 hours. After the reaction was completed, let it stand at room temperature to naturally evaporate the solvent until a dark red complex appeared. Filter and separate the solid product, and vacuum dry it at 40 °C for 12 hours for infrared spectrum analysis. The binding mode of Y and Cu 2+ was studied by infrared spectrum. As Figure 15 shown, the stretching vibration absorption peaks of O - H and C=N of Y were at 3433 cm -1 and 1632 cm -1 ​appears at the position. With Cu 2+ After complexation, the O-H stretching vibration peak shifts to 3500 cm -1 , the C=N broadens and shifts to 1604 cm -1 at the position. In addition, a new peak appears at 1393 cm -1 , and it is speculated that NO3 2- participates in the coordination.

[0073] The complexation ratio of Y with Cu 2+ is determined by Job's plot and mass spectrometry analysis. By the equimolar continuous variation method, as the mole fraction of Y increases, the fluorescence intensity of the system gradually increases. When the mole fraction of Y is 0.5, an inflection point appears linearly in the fluorescence, indicating that Y and Cu 2+ form a complex in a ratio of 1:1 ( Figure 11 ). The binding constant determined from the Benesi-Hilderbrand equation is 1.34×10 -3 M -1 ( Figure 12 ). In addition, in the presence of Cu 2+ , a proton peak appears at m / z 568.25 in the HR-MS spectrum of Y, which can be attributed to (calcd, m / z: 568.09)( Figure 13 ), further confirming the formation of the 1:1 Y-Cu 2+ complex.

[0074] The binding mode of Y-Cu 2+ was further studied in deuterated DMSO ( Figure 14 ). The peak at 13.99 ppm is the phenolic -OH signal peak (H1), and the peak at 8.68 ppm is the imine proton (H2) peak. When the solid Cu(NO3)2·3H2O is added, as the titration starts, the phenolic -OH signal peak at 13.99 ppm gradually disappears, indicating that the phenolic hydroxyl group of the probe Y participates in the coordination of Cu 2+ through the deprotonated oxygen atom. A significant change of 2.53 ppm downfield shift (8.68 ppm - 11.21 ppm) appears in the imine proton (H2) peak at 8.68 ppm with the gradual increase of copper ions, indicating that the imine N atom participates in the copper ion complexation. In addition, since the benzene rings where H3, H4, H 10 are located are connected to the functional groups participating in the coordination, resulting in a change in their electron clouds, and downfield shifts of 2.18 ppm (7.83 ppm - 10.01 ppm), 0.14 ppm (7.74 ppm - 7.88 ppm), and 0.5 ppm (6.88 ppm - 6.93 ppm) appear respectively. H 14There is also an offset of 0.94 ppm. Although the connection between the benzene ring (H5, H6, H7, H8, H9) and the directly coordinated atoms is relatively far, through the electron transfer of the conjugated system, the electron cloud density around it will still be affected, resulting in different degrees of chemical shift. Similarly, H 11 , H 12 The olefin group where it is located is also affected by the conjugated system, resulting in different degrees of chemical shift. From this, we speculate that the possible binding mode of Y and Cu 2+ is as shown in Figure 16 .

[0075] Example 4

[0076] Y-Cu 2+ Combined probe for the recognition and detection of S 2-

[0077] Copper ions (Cu 2+ ) and S 2- ions can form stable CuS (K sp = 6.3×10 -36 ). Therefore, based on the competitive substitution reaction, the Y-Cu 2+ complex as a combined probe has the potential for the detection of S 2+ . In a DMF-HEPES (1:9, v / v, 25 mM HEPES, pH 7.4) solution, Y and Cu 2- were mixed in a 1:1 ratio to form the Y-Cu 2+ complex. The fluorescence sensitivity of Y-Cu 2+ to S 2+ was investigated by fluorescence titration. As can be seen from Figure 17 , as the concentration of S 2- gradually increases, the fluorescence intensity of the system at 623 nm increases significantly, indicating that S 2- competes and substitutes Cu 2- from the Y-Cu 2+ complex and releases Y again. And it reaches the maximum value after adding 3-fold S 2+ . There is a good linear relationship between the fluorescence intensity and the concentration of S 2- in the range of 1 - 15 μM ( 2- ), and the correlation coefficient is 0.9958. Using the formula 3σ / s, the detection limit was calculated to be 2.13×10 Figure 18 M. -8

[0078] The sensing mechanism of Y3-Cu 2+ to S 2- was studied by UV-visible absorption spectroscopy. As shown in Figure 19 , Y-Cu 2+ ​​The solution has a maximum absorption peak at 494 nm. After adding 12 μM S 2- ions, the maximum absorption peak of the solution blue-shifts from 494 nm back to 425 nm, indicating that S 2- competes and substitutes Cu 2+ from the Y-Cu 2+ complex, releasing Y again.

[0079] As Figure 20 shown, the sensing performance of Y-Cu 2+ towards anions was investigated. Various anions such as SO3 2- , SO4 2- , Cl - , HPO4 2- , Br - , H2PO4 - , CO3 2- , HSO3 - , HCO3-, F - , NO3 - , I - and Ac - were separately added to the Y-Cu 2+ complex solution. It can be seen that after adding S 2- , a significant enhancement in fluorescence at 623 nm was observed, achieving a fluorescence recovery rate of 92.92% (fluorescence intensity increased from 316 to 1209). In contrast, there was no obvious change in the fluorescence intensity of the system after adding other anions. This result indicates that only the introduction of S 2- can effectively disrupt the complexation between the probe Y and Cu 2+ . In addition, even in the presence of other anions, it does not affect the detection of S 2- . The experimental results further confirm the excellent performance of Y-Cu 2+ in the selective detection of S 2- , providing strong support for its practical application in anion analysis.

[0080] Example 5

[0081] Detection of Cu 2+ and S 2- in water samples

[0082] The practical application performance of Y in detecting Cu 2+ and S 2- was investigated by detecting the water samples of the Fenhe River. The results are shown in Table 3. The probe can perform spike recovery detection on the concentrations of Cu 2+ and S 2- in tap water, and has a good recovery rate, indicating that this method can be used for the detection of Cu 2+ and S in actual water samples2- Detection

[0083] Table 3 Cu in the Fenhe River water sample 2+ and S 2- Ion spike recovery detection

[0084]

[0085] Example 6

[0086] Test strip experiment

[0087] After drying the probe test strip loaded with 5 μM Y, it was immersed in solutions containing different Cu 2+ concentrations (0 μM, 5 μM, 10 μM, 14 μM), and the color change of the test strip was recorded under 365 nm ultraviolet light. Similarly, the dried test strip loaded with Y-Cu 2+ complex was immersed in solutions with different S 2- concentrations (0 μM - 15 μM), and after drying, its color change was observed under 365 nm ultraviolet light.

[0088] Based on the good fluorescence performance of the probe and the characteristics of the color change of Cu 2+ and S 2- ions, a filter paper colorimetric experiment was further carried out to expand the application of Cu 2+ and S 2- detection. The dried paper strip attached with the probe Y was soaked in solutions with different concentrations of Cu 2+ and observed under an ultraviolet lamp after drying at room temperature. It can be found that as the Cu 2+ concentration increases, the color of the filter paper strip gradually changes from light red to purple under the ultraviolet lamp ( Figure 21 the upper line). In addition, the test strips prepared based on Y3-Cu 2+ showed obvious color changes when different concentrations of S 2- were added. Under 365 nm UV light, we saw that the color of the filter paper strip changed from purple back to light red ( Figure 21 the lower line). This experiment shows that the developed filter paper provides a cheap, convenient and simple method for the detection of Cu 2+ and S 2- ions.

[0089] Example 7

[0090] Detection of Cu using a smartphone 2+

[0091] An experimental setup for smartphone-assisted sensing was created. The sample was placed in a dark box to avoid light source interference, and a 365 nm (3 W) LED lamp was used as the excitation source. Fluorescence images were captured using a smartphone camera at night, and a dedicated application (Color Desk) was used to process the images to obtain the corresponding RGB values. As Figure 22 shown, the RGB values have a good linear relationship with the Y and Cu 2+ concentrations. It provides a simple method that can be completed without complex instruments for on-site detection of Cu 2+ .

[0092] Example 8

[0093] Toxicity experiment

[0094] To explore the potential bioanalytical applications of Y, the CCK-8 assay was used to measure the viability of probe cells after 24 hours of treatment. According to Figure 23 the results shown, after treatment with Y in the range of 0 - 50 μM, the cell viability was above 80%, indicating that Y has weak toxicity to cells.

[0095] Cell imaging experiment

[0096] Ovarian cancer cells A2780 were cultured in 1640 medium mixed with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin, and the incubator temperature was set at 37 °C, and the CO2 concentration was maintained at 5%. Then, A2780 cells were seeded in a culture dish and allowed to attach to the surface of the dish within 24 hours, and then the unattached cells were removed. Next, 20 μM Y was added to the culture dish and incubated for 10 minutes as a blank control experiment.

[0097] For exogenous Cu 2+ and S 2- , first, different concentrations of Cu 2+ (10 μM, 20 μM, 22 μM) were added to the medium and cultured for 10 min, then 20 μM Y was added to the medium and incubated with A2780 cells for 10 min. The medium was taken out and washed 3 times with PBS. Using PBS as the background, the fluorescence imaging changes of different concentrations of Cu 2+ under a confocal microscope (λex = 561 nm, 63× objective) were observed. To verify the detection of S 2+ in cells by the Y-Cu 2- combination probe, Y (20 μM) and Cu 2+ (22 μM) were cultured for 10 min, washed three times with PBS, and different concentrations of S 2- were added to the medium and cultured for 10 min, and the fluorescence imaging changes under confocal (λex = 561 nm, 63× objective) were observed.

[0098] In endogenous Cu 2+ and S 2- In cell imaging, first add 100 μM ascorbic acid to the culture medium and incubate for 10 min, then add different concentrations of Cu 2+ (10 μM, 20 μM, 22 μM) and incubate for 10 min, and perform fluorescence imaging under confocal (λex = 561 nm, 63× objective). For the S 2- cell imaging experiment, first add 20 μM Y and 22 μM Cu 2+ to one group. After incubating for 10 min, add 100 μM SNP solution, continue to incubate for 10 min, and finally complete the imaging observation.

[0099] Based on the spectral response of Y, the ability of Y to perform fluorescence imaging was further investigated using a confocal fluorescence microscope. Y (20 μM) was used for the Cu 2+ and S 2- imaging study in A2780 live cells. First, when the cells were incubated with Y (20 μM) for 10 min, significant strong fluorescence was observed in the red channel ( Figure 24 a). Then, when different concentrations of Cu 2+ were added and treated for 10 min, it could be seen that the fluorescence gradually quenched in the red channel ( Figure 24 b-d). After that, different concentrations of S 2- were added, and the red fluorescence gradually recovered ( Figure 25 c-e). The cells maintained good cell morphology throughout the process. These experiments indicate that Y has good cell compatibility and can be used for the cellular sensing of Cu 2+ and S 2- in cells.

[0100] It is reported in the literature that ascorbate can promote the uptake of ceruloplasmin in plasma, and ceruloplasmin is a reducing agent that can cause a shift in the Cu 2+ / Cu + redox reaction equilibrium and reduce the content of Cu 2+ . Therefore, ascorbic acid was selected to investigate the change in the content of endogenous Cu 2+ in cells by probe Y. The experimental results show that the cell samples treated with ascorbic acid ( Figure 24 e) have stronger fluorescence than the samples not treated with ascorbic acid ( Figure 24 a), indicating that the content of Cu 2+ in the cells treated with ascorbic acid is lower than that in the untreated cells. Similarly, similar phenomena were also observed in Figure 24 bcd and 24fgh. It shows that Y can detect the change in the content of intracellular Cu 2+ .

[0101] Cystathionine γ-lyase (CSE) is a key enzyme for H2S synthesis. NO can stimulate an increase in the content of CSE, thereby increasing the content of H2S. Therefore, we stimulated the production of S in cells by adding sodium nitroprusside (SNP), a NO donor. 2- Cells cultured in a medium coexisting with Y and Cu 2+ showed no obvious fluorescence. After adding 100 μM SNP, obvious red fluorescence ([ Figure 25 g) could be observed, indicating that Y-Cu 2+ could be used for intracellular endogenous S 2- imaging. The above experiments showed that Y could monitor the changes in the contents of intracellular / exogenous Cu 2+ and S. 2- ​

Claims

1. An isophorone derivative Y, characterized in that, Its structural formula is: 。 2. The preparation method of an isophorone derivative Y according to claim 1, characterized in that, Weigh 5-[(3-dicyanomethylene-5,5-dimethyl)cyclohex-1-enyl]salicylaldehyde into a two-necked flask, dissolve it by reflux stirring with ethanol at 80 °C. After complete dissolution, add benzylamine and reflux stir for 10 hours. Cool to room temperature, place the resulting liquid in the refrigerator and store it at 4 °C for two days. After the liquid precipitates solids, collect them by vacuum filtration, and wash the crude product three times with cold ethanol and methanol in turn to obtain the isophorone derivative Y.

3. The preparation method of an isophorone derivative Y according to claim 1, characterized in that, The molar ratio of the 5-[(3-dicyanomethylene-5,5-dimethyl)cyclohex-1-enyl]salicylaldehyde to benzylamine is 1:1, and the molar volume ratio of benzylamine to ethanol is 3 mmol:200 mL.

4. Application of isophorone derivative Y in Cu 2+ detection.

5. The application according to claim 4, wherein After drying the probe test strip loaded with 5 µM Y, it was immersed in solutions containing different Cu 2+ concentrations, and the color change of the test strip was recorded under 365 nm ultraviolet light irradiation. As the Cu 2+ concentration increased, the color of the filter paper strip gradually changed from light red to purple under ultraviolet light.

6. The application according to claim 4, characterized in that Place the test sample containing Cu 2+ in a dark box to avoid light source interference, use a 365 nm 3 W LED lamp as the excitation source, capture the fluorescence image using a smartphone camera at night, and process the image using the application Color Desk program to obtain the corresponding RGB values. The RGB values show a good linear relationship with the Y and Cu 2+ concentrations.

7. Application of isophorone derivative Y in intracellular / extracellular Cu imaging in cells, characterized in that, 2+ It includes the following steps: When the cells were first incubated with 20 µM of Y for 10 min, significant strong fluorescence was observed in the red channel, and then different concentrations of Cu were added respectively. 2+ When treated for 10 min, as the concentration of Cu 2+ increased, the fluorescence in the red channel gradually quenched. ​ 8. Application of isophorone derivative Y-Cu 2+ complex in S 2- detection 9. The application according to claim 8, wherein Immerse the dried test strip of the Y-Cu 2+ complex in solutions of different S 2- concentrations. After drying, observe the color change under 365 nm ultraviolet light. As the S 2- concentration increases, the color of the filter paper strip changes from purple back to light red.

10. Application of isophorone derivative Y-Cu 2+ complex in endogenous / exogenous S 2- imaging in cells, characterized in that Incubate the cells with 20 µM of Y and 22 µM of Cu 2+ for 10 min, and then add different concentrations of S to the culture medium 2- Incubate for 10 min. The fluorescence imaging changes as follows: as the concentration of S 2- gradually increases, the fluorescence intensity of the Y-Cu 2+ complex at 623 nm increases significantly, showing a good linear relationship with S 2- in the concentration range of 1 - 15 µM, with R 2 = 0.9958. Therefore, S can be quantitatively detected by fluorescence spectrometry 2- .

Images