A cadmium ion detection reagent, a preparation method and application thereof

Abstract

This invention provides a cadmium ion detection reagent, its preparation method, and its application, to solve the problems of cadmium ion detection in the prior art. 2+ Ion detection suffers from high costs, technical complexity, and time consumption, and does not allow for high-throughput analysis. This invention addresses the Cd... 2+ The structure of the ion detection reagent is as follows: ; its preparation method includes the following steps: (1) Add phenanthroline aldehyde and 2-hydroxyacetophenone to a solvent and stir evenly, then add alkali and catalyst, react at room temperature, and then purify to obtain the target product. The cadmium ion detection reagent of the present invention can qualitatively and quantitatively detect cadmium ions, with a detection limit of 2.12 × 10⁻⁶. ‑7 M.

Description

Technical Field

[0001] This invention belongs to the field of metal ion detection technology, and particularly relates to a cadmium ion detection reagent, its preparation method, and its application. Background Technology

[0002] Cadmium ions pose extremely serious and persistent harm to the human body. Its toxicity primarily manifests through long-term, low-dose exposure, accumulating mainly in the kidneys and liver after entering the body, with a biological half-life of 10-30 years. The most typical harm of chronic cadmium exposure is irreversible renal tubular damage, manifested as low-molecular-weight proteinuria, severely affecting renal reabsorption function and ultimately leading to kidney failure. Cadmium also severely interferes with calcium and phosphorus metabolism, replacing calcium in bones, leading to decreased bone mineral density, osteoporosis, and osteomalacia; the historically infamous "Itai-itai disease" in Japan is a typical example. Furthermore, cadmium has a proven carcinogenic effect, classified as a Group 1 human carcinogen by the International Agency for Research on Cancer (IARC), with long-term exposure significantly increasing the risk of lung cancer, prostate cancer, and other diseases. Studies have also shown that cadmium exposure is closely related to cardiovascular disease, endocrine disorders (such as affecting insulin secretion), and damage to the nervous system. Even low-concentration, long-term exposure can adversely affect children's neurodevelopment and adult reproductive health. Therefore, establishing efficient and sensitive methods for detecting cadmium ions is crucial for preventing early exposure, assessing health risks, and guiding interventions. Thus, developing methods for detecting Cd in biological and environmental analysis is essential. 2+ Fluorescent probes are very important. Although there are some fluorescent probes used for the detection of Cd... 2+ Ions, but for Cd 2+ Designing ions with low toxicity, high affinity, and high selectivity for other metal ions remains a challenging task.

[0003] Currently, traditional methods such as voltammetry, potentiometry, atomic absorption spectrometry (AAS), inductively coupled plasma mass spectrometry (ICP-MS), and atomic emission spectrometry (AES) have been used to detect metal ions. However, these methods are costly, technically complex, and time-consuming, preventing high-throughput analysis. Fluorescence sensing methods for metal ion detection offer advantages such as speed, simplicity, and low cost. Therefore, developing a highly sensitive and selective fluorescence detection method for cadmium ions that can operate under weakly acidic conditions is of significant practical importance. Summary of the Invention

[0004] The purpose of this invention is to provide a cadmium ion detection reagent, its preparation method, and its application, in order to solve the problems of cadmium ion detection in the prior art. 2+ Ion detection is characterized by high cost, technical complexity and time consumption, and the inability to perform high-throughput analysis.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A cadmium ion detection reagent, the structure of which is shown below: .

[0006] Furthermore, the synthetic route for the cadmium ion detection reagent is as follows: .

[0007] Another objective of this invention is to provide a method for preparing a cadmium ion detection reagent, which involves adding an aldehyde and a 2-hydroxyacetophenone derivative to a solvent, then adding an alkali and a catalyst, reacting at room temperature, and finally purifying the reagent.

[0008] Furthermore, the 2-hydroxyacetophenone derivative is at least one selected from 3-bromoacetophenone, 2'-bromoacetophenone, 2-methyl-4-hydroxyacetophenone, o-aminoacetophenone, 3'-fluoroacetophenone, m-chloroacetophenone, 4-fluoro-2-hydroxyacetophenone, and 4-ethoxy-2-hydroxyacetophenone.

[0009] Furthermore, the solvent is at least one selected from anhydrous methanol, anhydrous ethanol, ethylene glycol, isopropanol, acetone, butanone, tetrahydrofuran, and dioxane.

[0010] Furthermore, the alkali is at least one selected from sodium hydroxide, potassium hydroxide, barium hydroxide, lithium hydroxide, sodium ethoxide, and triethylamine.

[0011] Furthermore, the catalyst is at least one selected from 4-dimethylaminopyridine, 4-pyrrolylpyridine, N-oxypyridine, 4-hydroxypyridine, 4-aminopyridine, methylpyridine, nitropyridine, and ethylpyridine.

[0012] Further purification is performed as follows: the reaction solution is poured into cold water (0℃~10℃) to precipitate solids, filtered to obtain a yellow solid, the precipitate is washed with washing solution, and the solvent is removed under vacuum to obtain the target product.

[0013] Another object of the present invention is to provide an application of a cadmium ion detection reagent for the qualitative and quantitative detection of cadmium ions, with a detection limit as low as 2.12 × 10⁻⁶. -7 M.

[0014] Furthermore, the cadmium ion detection reagent of the present invention is used for the detection of cadmium ions in biological cells. The detection method is as follows: the biological cells are cultured in PBS buffer containing the cadmium ion detection reagent, the cadmium ion detection reagent outside the biological cells is washed with PBS buffer, the cells are irradiated with UV light with a wavelength of 365 nm, and the color change is observed using an optical microscope.

[0015] Mechanism: Cd 2+The fluorescence enhancement effect is related to the complexation of the N atom on the phenanthroline ring and the hydroxyl groups on the two ortho-hydroxyacetophenones, exhibiting a significant fluorescence enhancement effect (CHEF). A semi-rigid cavity is formed in the compound structure, perfectly matching the target metal ion. The CHEF effect is due to the host complexation of Cd. 2+ Subsequently, the PET process is inhibited, specifically as follows: Figure 6 As shown.

[0016] The advantages of this invention are: 1. This invention employs fluorescence sensing technology, which has advantages such as simplicity, fast response speed, good selectivity, high sensitivity, convenient operation, and real-time detection. It introduces a fluorophore containing phenanthroline for the detection of Cd in organic solutions and aqueous systems. 2+ High sensitivity and selective recognition of ions; the synthesized fluorescent probe almost meets the key characteristics that intracellular fluorescent probes should have, with an excitation wavelength of 340 nm to prevent ultraviolet-induced cell damage; and an emission wavelength close to 500 nm to avoid the cell's own natural fluorescence, so as to facilitate cell imaging experiments using a fluorescence microscope. 2. The cadmium ion reagent provided by this invention can qualitatively and quantitatively detect cadmium ions in aqueous solution, and has promising applications in the qualitative and quantitative detection of cadmium ions, with a detection limit as low as 2.12 × 10⁻⁶. -7 M; and the preparation process of the cadmium ion detection reagent of the present invention is simple, with a yield of 98.1%. Attached Figure Description

[0017] Figure 1 For the present invention Cd 2+ A comparison of fluorescence of ions and other metal ion testing reagents.

[0018] Figure 2 For the present invention Cd 2+ A schematic diagram showing the fluorescence data of each group of solutions containing different metal ions after adding ion detection reagents.

[0019] Figure 3 The present invention provides a cadmium ion detection reagent for detecting intracellular Cd in HeLa cells. 2+ A schematic diagram.

[0020] Figure 4 The present invention uses cadmium ion detection reagent and Cd 2+ Fluorescence images of HeLa cells incubated. In the images, cells were incubated with 20 μM Cd for 30 min (a and b), followed by further incubation with 20 μM Cd. 2+ Incubate for 30 min (c and d); observe the left images (a and c) using an optical microscope and take the right images (b and d) using a fluorescence microscope.

[0021] Figure 5 The invention under natural light and the invention Cd 2+ Color changes of ions under UV light (365 nm).

[0022] Figure 6 This is a diagram illustrating the reaction mechanism of the present invention. Detailed Implementation

[0023] Example 1 A cadmium ion detection reagent, the synthesis route of which is as follows: The specific synthesis process of the target product is as follows: 1.1 mmol of 2-hydroxyacetophenone and 1 mmol of phenanthroline aldehyde were added to anhydrous CH3OH (10 mL) and 5% pyridine solution. Sodium hydroxide solution (2.5 M) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was poured into cold water and filtered. The resulting yellow precipitate was washed three times with cold CH3OH (8 mL). The solvent was removed under vacuum to obtain the intermediate product with a yield of 98.1%.

[0024] Characterization results of the target product: 1) Infrared spectroscopy measurement: Potassium bromide tablets were used in the 400-4000 cm⁻¹ range. -1 The results were determined using a Nicolet Magna 750 Fourier transform infrared spectrometer (USA). The main infrared absorption peaks of the obtained compounds were: 3355, 2977, 1683, 1612, 1451, and 1167 cm⁻¹. -1 .

[0025] 2) Nuclear magnetic resonance hydrogen spectroscopy determination: The proton NMR spectra of the obtained compound were determined using deuterated chloroform as solvent and tetramethylsilane as internal standard, and a Bruker 400 NMR spectrometer. The proton NMR spectra of the obtained compound are as follows: 1 H NMR (DMSO-d6) δ: 9.47 (s, 2H), 8.72(d, J = 8.0Hz, 2H), 8.67 (d, J = 8.4 Hz, 2H), 8.43(d, J = 8.0 Hz, 2H), 8.16 (s, 2H), 7.50(d, J = 9.2 Hz, 2H), 7.20(t, J = 8.4 Hz, 2H), 6.99 (d, J = 8.1 Hz, 2H), 6.92(t, J = 8.1 Hz, 2H), δ 5.2 - 5.4 (m, 2H).

[0026] 3) Carbon NMR spectroscopy: The carbon NMR spectrum of the obtained compound was determined using deuterated dimethyl sulfoxide as solvent and tetramethylsilane as internal standard, and analyzed by a Bruker 400 NMR spectrometer. The carbon NMR spectrum of the obtained compound is as follows: 13 C NMR δ189.51, 172.56, 165.82, 145.55, 135.50, 129.26, 128.18, 121.42, 118.17, 115.42, 60.39, 55.87, 30.45, 20.54.

[0027] 4) Elemental analysis: Elemental analysis was performed using a PE2400-11 elemental analyzer from PE Corporation, USA. The elemental analysis values ​​of the obtained compounds are: Found C: 76.27, H: 4.27, N: 5.93, O: 13.53.

[0028] 5) Mass spectrometry determination: Mass spectrometry was performed using a Bruker DataAnalysis mass spectrometer: The mass spectrum of the obtained compound was: [M+Na]+: 495.13; (theoretical value), found: 495.15.

[0029] Application of the target product in the detection of cadmium ions: The cadmium ion detection reagent obtained in Example 1 was prepared to a concentration of 3.0 × 10⁻⁶. -5 A DMSO solution with a concentration of mmol / L. The solutions containing (Li) were compared with those containing (Li) + Na + K + Ag + Zn 2+ Mg 2+ Ba 2+ Ca 2+ Mn 2+ Pb 2+ Hg 2+ Ni 2+ Cd 2+ Co 2+ Cu 2+ Fe 2+ Cr 3+ Fe 3+ Al 3+ and Eu 3+ The DMSO solution (etc.) was used as the detection target. The results showed that: Adding Cd 2+ When ions are added, the fluorescence of the detection reagent is enhanced, while the fluorescence of the test reagent remains almost unchanged when other metal ions are added, such as... Figure 1 As shown. In the experiment, 3mC Ld was taken. 2+Ion detection reagents were added to a cuvette, followed by the addition of solutions containing different types and concentrations of metal ions, which were then thoroughly mixed. The fluorescence data of each solution was then measured. Figure 2 As shown. The detection limit, calculated through fluorescence titration and fitting, is as low as 2.12 × 10⁻⁶. -7 M ( Figure 3 The cadmium ion detection reagent of the present invention can detect cadmium ions qualitatively and quantitatively.

[0030] The above spectroscopic experimental studies show that the cadmium ion detection reagent can specifically and sensitively identify Cd. 2+ Therefore, in order to further expand the application scope of cadmium ion detection reagents, laser confocal microscopy was used to detect Cd ions in human cervical cancer cell line HeLa. 2+ Conduct testing.

[0031] This embodiment investigated the effect of 20 μM Cd on Cd in HeLa cells. 2+ Ion recognition performance. HeLa cells were cultured in PBS buffer containing cadmium ion detection reagent (10 μM) for 30 min. The cadmium ion detection reagent outside the cells was washed off with the same concentration of PBS buffer, followed by Cd... 2+ Reculture cells in 20 μM PBS buffer for 30 min. Figure 4 As shown, after adding Cd 2+ Before ion detection, an optical microscope captured no blue fluorescence, indicating that the cadmium ion detection reagent can penetrate the cell wall and enter the cell. (Adding Cd...) 2+ After ionization, a significant enhancement of fluorescence was observed in optical microscopy. These fluorescence images obtained through optical microscopy demonstrate the great potential of the cadmium ion detection reagent for basic biological research.

[0032] Figure 5 The cadmium ion detection reagent of the present invention has an excitation wavelength of about 365 nm, which can prevent ultraviolet-induced cell damage.

[0033] In some other embodiments, 3-bromoacetophenone, 2'-bromoacetophenone, 2-methyl-4-hydroxyacetophenone, o-aminoacetophenone, 3'-fluoroacetophenone, m-chloroacetophenone, 4-fluoro-2-hydroxyacetophenone, and 4-ethoxy-2-hydroxyacetophenone were used instead of 2-hydroxyacetophenone in Example 1.

[0034] A cadmium ion detection reagent with the corresponding chemical formula structure was obtained. It was measured according to the characterization method of Example 1 and applied in an application test. The results were similar to those of Example 1.

[0035] In some other embodiments, anhydrous methanol in Example 1 was replaced with anhydrous ethanol, ethylene glycol, isopropanol, acetone, butanone, tetrahydrofuran, or dioxane; sodium hydroxide in Example 1 was replaced with potassium hydroxide, barium hydroxide, lithium hydroxide, sodium ethoxide, or triethylamine; and pyridine in Example 1 was replaced with 4-dimethylaminopyridine, 4-pyrrolidinylpyridine, N-pyridine oxide, 4-hydroxypyridine, 4-aminopyridine, methylpyridine, nitropyridine, or ethylpyridine. In all these cases, the cadmium ion detection reagent of Example 1 was obtained, and the yield was close to that of Example 1. Specific embodiments are as follows.

[0036] Example 2 1.1 mmol of 3-bromoacetophenone and 1 mmol of phenanthroline aldehyde were added to anhydrous CH3OH (10 mL) and a 5% pyridine solution. Sodium hydroxide solution (2.5 M) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was then poured into cold water, filtered, and the resulting yellow precipitate was washed three times with cold CH3OH (8 mL). The solvent was removed under vacuum to obtain the product, with a yield of 97.1%. mp: 210.7–211.4 °C. Infrared spectrum (cm²) -1 ): 3347, 2979, 1682, 1590, 1522, 1496, 1451, 1166.

[0037] Example 3 1.1 mmol of 2'-bromoacetophenone and 1 mmol of phenanthroline aldehyde were added to anhydrous CH3OH (10 mL) and a 5% pyridine solution. Sodium hydroxide solution (2.5 M) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was then poured into cold water, filtered, and the resulting yellow precipitate was washed three times with cold CH3OH (8 mL). The solvent was removed under vacuum to obtain the product, with a yield of 97.5%. mp: 205-206℃. Infrared spectrum (cm²) -1 ): 3355, 2977, 1683, 1612, 1493, 1451, 1167.

[0038] Example 4 1.1 mmol of o-aminoacetophenone and 1 mmol of phenanthroline aldehyde were added to anhydrous CH3OH (10 mL) and a 5% pyridine solution. Sodium hydroxide solution (2.5 M) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was then poured into cold water, filtered, and the resulting yellow precipitate was washed three times with cold CH3OH (8 mL). The solvent was removed under vacuum to obtain the product, with a yield of 96.8%. mp: 199.7–201.8℃. Infrared spectrum (cm²) -1): 3351, 2983, 1685, 1621, 1498, 1454, 1159.

[0039] Example 5 1.1 mmol of 3'-fluoroacetophenone, 1.1 mmol of o-aminoacetophenone, and 1 mmol of phenanthroline aldehyde were added to anhydrous CH3OH (10 mL) and a 5% pyridine solution. Sodium hydroxide solution (2.5 M) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was then poured into cold water, filtered, and the resulting yellow precipitate was washed three times with cold CH3OH (8 mL). The solvent was removed under vacuum to obtain the product, with a yield of 96.4%. mp: 226-227℃. Infrared spectrum (cm²) -1 ): 3352, 2933, 1668, 1640, 1461, 1442, 1182.

[0040] Example 6 1.1 mmol of m-chloroacetophenone and 1 mmol of phenanthroline aldehyde were added to anhydrous CH3OH (10 mL) and a 5% pyridine solution. Sodium hydroxide solution (2.5 M) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was then poured into cold water, filtered, and the resulting yellow precipitate was washed three times with cold CH3OH (8 mL). The solvent was removed under vacuum to obtain the product, with a yield of 95.9%. mp: 226-227℃. Infrared spectrum (cm²) -1 ): 3348, 2991, 1681, 1631, 1509, 1432, 1147.

[0041] Example 7 1.1 mmol of 4-ethoxy-2-hydroxyacetophenone and 1 mmol of phenanthroline aldehyde were added to 10 mL of anhydrous CH3OH and a 5% pyridine solution. Sodium hydroxide solution (2.5 M) was slowly added dropwise. After the addition was complete, the mixture was stirred at room temperature for 3 h. The reaction solution was then poured into cold water, filtered, and the resulting yellow precipitate was washed three times with cold CH3OH (8 mL). The solvent was removed under vacuum to obtain the product, with a yield of 94.7%. mp: 219-220℃. Infrared spectrum (cm²) -1 ): 3352, 2987, 1659, 1631, 1484, 1429, 1167.

Claims

1. A cadmium ion detection reagent, characterized in that: The structure of the detection reagent is shown below: 。 2. The cadmium ion detection reagent as described in claim 1, characterized in that, Its synthetic route is as follows: 。 3. The method for preparing the cadmium ion detection reagent according to any one of claims 1 or 2, characterized in that: The aldehyde and 2-hydroxyacetophenone derivative are added to a solvent, followed by the addition of an alkali and a catalyst. The mixture is reacted at room temperature and then purified.

4. The preparation method of the cadmium ion detection reagent as described in claim 3, characterized in that: The 2-hydroxyacetophenone derivative is at least one of 3-bromoacetophenone, 2'-bromoacetophenone, 2-methyl-4-hydroxyacetophenone, o-aminoacetophenone, 3'-fluoroacetophenone, m-chloroacetophenone, 4-fluoro-2-hydroxyacetophenone, and 4-ethoxy-2-hydroxyacetophenone.

5. The method for preparing the cadmium ion detection reagent as described in claim 3, characterized in that: The solvent is at least one of anhydrous methanol, anhydrous ethanol, ethylene glycol, isopropanol, acetone, butanone, tetrahydrofuran, and dioxane.

6. The method for preparing the cadmium ion detection reagent as described in claim 3, characterized in that: The alkali is at least one selected from sodium hydroxide, potassium hydroxide, barium hydroxide, lithium hydroxide, sodium ethoxide, and triethylamine.

7. The method for preparing the cadmium ion detection reagent as described in claim 3, characterized in that: The catalyst is at least one selected from 4-dimethylaminopyridine, 4-pyrrolylpyridine, N-oxypyridine, 4-hydroxypyridine, 4-aminopyridine, methylpyridine, nitropyridine, and ethylpyridine.

8. The method for preparing the cadmium ion detection reagent as described in claim 3, characterized in that: The purification method is as follows: the reaction solution is poured into water at 0~10℃, solids are precipitated, filtered to obtain a yellow solid, the precipitate is washed with washing solution, and the solvent is removed under vacuum to obtain the target product.

9. The application of the cadmium ion detection reagent as described in any one of claims 1 or 2, characterized in that: This test reagent is used for qualitative and quantitative detection of cadmium ions, with a detection limit as low as 2.12 × 10⁻⁶. -7 M.

10. The application of the cadmium ion detection reagent as described in any one of claims 1 or 2, characterized in that: It is used in the preparation of cadmium ion detection products for biological cells.

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