Small-molecule fluorescent probe and application thereof in nickel ion detection

Abstract

The invention discloses a small-molecule fluorescent probe and application thereof in nickel ion detection. Wherein the small-molecule fluorescent probe is a coupling derivative based on dicyanoisophorone (DCI) and hydroxyphenyl quinazolinone (HPQ), the molecular formula of the small-molecule fluorescent probe is C35H29ClN4O6, and the structure of the small-molecule fluorescent probe is as follows: # imgabs0 #. Compared with the traditional metal ion detection technology, the micromolecular fluorescent probe provided by the invention has the advantages that the reaction is rapid, the operation is simple and convenient, professional technicians or expensive equipment is not needed, the detection cost is greatly reduced, and the efficiency of the detection process is improved.

Description

Technical Field

[0001] The present invention belongs to the field of organic chemistry, and particularly relates to a small molecule fluorescent probe and its application in nickel ion detection. Background Art

[0002] With the widespread use of e-cigarettes, regulatory agencies and public health organizations have increasingly focused on the heavy metal ions that may be contained in the aerosols produced by e-cigarettes, such as nickel, because it may pose a risk to public health. Existing nickel ion detection methods usually involve complex sample processing and high-cost equipment, which pose challenges to the formulation of e-cigarette product standards and the monitoring of the safety of e-cigarettes on the market. Summary of the Invention

[0003] In view of the deficiencies of the above-mentioned existing technologies, the present invention provides a small molecule fluorescent probe and its application in nickel ion detection. Compared with traditional metal ion detection technologies, the small molecule fluorescent probe of the present invention not only reacts quickly, but also is easy to operate, without the need for professional technicians or expensive equipment, greatly reducing the detection cost and improving the efficiency of the detection process.

[0004] The small molecule fluorescent probe of the present invention is a coupling derivative based on dicyanoisophorone (DCI) and hydroxyphenylquinazolinone (HPQ), with the molecular formula C 35 H 29 ClN 4 O 6 , and its structure is shown as follows:

[0005]

[0006] The preparation method of the small molecule fluorescent probe of the present invention includes the following steps:

[0007] Step 1: First, add (3,5,5-trimethylcyclohex-2-enylidene) malononitrile (1.5 g, 8.05 mmol), p-hydroxybenzaldehyde (1.8 g, 14.74 mmol), piperidine (1.0 mL) and anhydrous ethanol to a single-necked round-bottom flask equipped with a condenser, heat to 85 °C and stir continuously for 5 hours, and monitor the reaction progress by thin-layer chromatography; after the reaction is completed, evaporate the solvent to obtain a crude product, and obtain pure compound i (orange fluorescent solid, yield 50%) by column chromatography using pure dichloromethane as the eluent.

[0008] Step 2: Dissolve the obtained (3,5,5-trimethylcyclohex-2-enylidene) malononitrile (0.5 g, 1.72 mmol) and hexamethylenetetramine (0.56 g, 3.99 mmol) in trifluoroacetic acid (5.0 mL), and reflux the reaction at 85 °C; after the reaction is completed, pour the reactant into 100 g of ice water and add 2.0 mL of hydrochloric acid, filter under reduced pressure and wash the dried solid with cold water, and further purify by column chromatography using pure dichloromethane to obtain pure compound ii (yellow solid, yield 80%).

[0009] Step 3: Dissolve compound ii (0.5 g, 1.57 mmol) in ethanol (15 mL) and dichloromethane (5 mL) to obtain a yellow solution, add 2-amino-5-chlorobenzamide (0.6 g, 3.51 mmol) at room temperature, heat to 95 °C and reflux for 30 minutes, then add p-toluenesulfonic acid monohydrate (0.02 equivalent), and continue to reflux for 2 hours; cool the resulting red suspension to room temperature, add DDQ (1.01 equivalent) in portions, stir overnight at room temperature, filter the precipitate, wash it three times with absolute ethanol and twice with dichloromethane, and dry to obtain compound iii (dark red powder, yield 42.5%).

[0010] Step 4: React compound iii and allyl chloroformate in dry tetrahydrofuran under basic conditions to prepare probe W11Ni. Dissolve compound iii in dry tetrahydrofuran, add diisopropylethylamine and stir, then cool in an ice bath for 5 minutes, and slowly add allyl chloroformate. Stir overnight at room temperature, extract the solution and rotary evaporate the solvent to dryness under reduced pressure. Separate the crude product by column chromatography using a mixture of petroleum ether and ethyl acetate (volume ratio 4:1) to obtain purified probe W11Ni.

[0011] The synthetic route is as follows:

[0012]

[0013] Application of the small molecule fluorescent probe of the present invention in the preparation of a nickel ion detection reagent.

[0014] When the Ni 2+ concentration is in the range of 5 nM - 50 μM, the fluorescence intensity of the detection reagent at 485 nm shows a linear relationship with the Ni 2+ concentration.

[0015] The detection reagent can achieve the detection of the Ni 2+ content in e-cigarette aerosols.

[0016] The present invention provides a detection method that is fast, accurate, and cost-effective. This method uses small molecule fluorescent probes to detect nickel ions in e-cigarette aerosols with high sensitivity and high selectivity. The design of the probe ensures that even in complex samples containing other ions, the concentration of nickel ions can be specifically identified and determined, which is crucial for evaluating whether e-cigarette products meet safety standards. In addition, the use of this fluorescent probe provides a tool for e-cigarette manufacturers to comply with national and international safety standards, helping them monitor and adjust the nickel content in real-time during the product design and quality control processes.

[0017] The fluorescence probe detection method of the present invention is fast, sensitive, and efficient, and can be widely applied to the quantitative detection of heavy metal nickel ions in fields such as e-cigarettes, environmental monitoring, and industrial wastewater, providing a simple, low-cost, and efficient detection tool for related industries.

[0018] In summary, the small molecule fluorescent probe of the present invention provides important technical support for the setting and implementation of regulatory standards in the e-cigarette industry, helps to promote the development of e-cigarette products towards a safer and more transparent direction, and safeguards the health and safety of consumers. Brief Description of the Drawings

[0019] Figure 1 is the mass spectrum of the small molecule probe W11Ni.

[0020] Figure 2 is the original ultraviolet absorption spectrum of the small molecule probe and the ultraviolet absorption spectrum after the addition of nickel ions with a response.

[0021] Figure 3 is the linear relationship curve between the fluorescence intensity of the small molecule probe and the concentration of nickel ions.

[0022] Figure 4 is the color change when detecting nickel ions in e-cigarette aerosols with the small molecule probe.

[0023] Figure 5 is the comparison data of interfering ions and nickel ions when the small molecule probe detects multiple interfering ions. Compared with the interfering ions, nickel ions show a 20-fold signal response. Detailed Description of the Embodiments

[0024] Description of the Embodiment: This embodiment describes a fluorescence probe detection system named Ni 2+ Test for quantitatively analyzing the concentration of nickel ions in e-cigarette aerosols or other samples. This detection system is designed to detect nickel ions quickly and accurately and is suitable for research and industrial quality control.

[0025] Example 1: Preparation of the Small Molecule Probe W11Ni

[0026] 1. First, add (3,5,5 - trimethylcyclohex - 2 - enylidene) malononitrile (1.5 g, 8.05 mmol), p - hydroxybenzaldehyde (1.8 g, 14.74 mmol), piperidine (1.0 mL) and anhydrous ethanol into a single - necked round - bottom flask equipped with a condenser. Slowly raise the reaction temperature of the mixture to 85 °C and stir continuously for 5 hours. Monitor the reaction progress by thin - layer chromatography. After the reaction is completed, evaporate the solvent to obtain the crude product. Through column chromatography, using pure dichloromethane as the eluent, pure compound i (orange - fluorescent solid, yield 50%) is obtained.

[0027] 2. Dissolve the obtained (3,5,5 - trimethylcyclohex - 2 - enylidene) malononitrile (0.5 g, 1.72 mmol) and hexamethylenetetramine (0.56 g, 3.99 mmol) in trifluoroacetic acid (5.0 mL), and reflux at 85 °C. After completion, pour the reactant into 100 g of ice water and add 2.0 mL of hydrochloric acid. Filter under reduced pressure and wash the dried solid with cold water. Further purify by column chromatography using pure dichloromethane to obtain pure compound ii (yellow solid, yield 80%).

[0028] 3. Prepare a yellow solution by dissolving compound ii (0.5 g, 1.57 mmol) in ethanol (15 mL) and dichloromethane (5 mL). Add 2 - amino - 5 - chlorobenzamide (0.6 g, 3.51 mmol) at room temperature. After heating to 95 °C and refluxing for 30 minutes, add p - toluenesulfonic acid monohydrate (0.02 equivalent), and continue refluxing for 2 hours. Cool the resulting red suspension to room temperature, add DDQ (1.01 equivalent) in batches, and stir overnight at room temperature. Filter the precipitate, wash it three times with absolute ethanol and twice with dichloromethane, and dry to obtain compound iii (dark red powder, yield 42.5%).

[0029] 4. React compound iii (100 mg, 0.213 mmol) and allyl chloroformate (272.3 mg, 2.556 mmol) in 10 mL of dry tetrahydrofuran under basic conditions to prepare probe W11Ni. Dissolve compound iii in dry tetrahydrofuran, add diisopropylethylamine (82.6 mg, 0.639 mmol), stir, then cool in an ice bath for 5 minutes, and slowly add allyl chloroformate. Stir overnight at room temperature, extract the solution and rotary evaporate the solvent to dryness under reduced pressure. Separate the crude product by column chromatography using a mixture of petroleum ether and ethyl acetate (volume ratio 4:1) to obtain purified probe W11Ni (yellow powder, yield 58.5%).

[0030] Example 2: Fluorescence Detection

[0031] The nickel ion fluorescence probe detection method provided by the present invention can achieve efficient quantitative analysis of the nickel ion concentration in the analyte through the prepared Ni 2+ Test system. In the specific implementation process, first, according to the pre-prepared system, the analyte is added to the detection solution containing the W11Ni fluorescence probe, triphenylphosphine (PPh 3 ), sodium borohydride (NaBH 4 ), etc. The ratio of this solution is optimized to ensure the specific binding of nickel ions to the fluorescence probe and the resulting change in the fluorescence signal.

[0032] After the sample preparation is completed, a fluorescence spectrometer is used to detect the reaction system. The fluorescence spectrometer monitors the change in the fluorescence signal in the analyte solution in real time according to the characteristic excitation and emission wavelengths of the probe. When nickel ions bind to the probe, the fluorescence intensity changes, reflecting the nickel ion concentration in the sample.

[0033] To achieve quantitative analysis, the present invention uses a pre-constructed concentration standard curve. The standard curve consists of known-concentration nickel standard solutions and corresponding fluorescence intensity data points. By comparing the fluorescence intensity of the sample with the relationship of the standard curve, the nickel ion concentration in the analyte can be accurately calculated. During the construction and testing of the standard curve, the linear relationship between the fluorescence signal and the nickel ion concentration is fully considered, and through multiple calibrations and verifications, the high precision and high reliability of the measurement results are ensured. The response sensitivity of the fluorescence probe W11Ni to nickel ions reaches 5 nM (LOD).

[0034] Example 3: Detection of nickel ions using the Ni 2+ Test system fluorescence probe detection system

[0035] Configure the detection system:

[0036] Preparation of solvents and reagents: First, prepare a 50 mL methanol:water mixed solvent with a ratio of 8:2. Mix methanol and water according to the volume ratio and stir well to ensure uniform mixing. Triphenylphosphine (PPh 3 ) is configured in methanol to prepare a 10 mM stock solution. Take an appropriate amount of triphenylphosphine, dissolve it in methanol, and store it sealed. Sodium borohydride (NaBH 4 ) is configured in water and also prepared as a 10 mM stock solution. Take an appropriate amount of sodium borohydride, dissolve it in water, and store it sealed.

[0037] Preparation of the probe and nickel standard solution: Dissolve the compound W11Ni (molecular weight 636.18) in dimethyl sulfoxide (DMSO) to prepare a 10 mM stock solution. The nickel standard solution is prepared as 10 mM and diluted by dissolving nickel sulfate in an appropriate solvent to match the requirements in the test.

[0038] Before detection, appropriate amounts were taken from each stock solution and mixed to a volume of 1 mL to ensure that the working concentration of probe W11Ni was 20 μM. At the same time, ensure that the final concentrations of PPh 3 and NaBH 4 in the test system were both 100 μM. The prepared test system was placed in an appropriate fluorescence spectrometer, and the settings of the spectrometer were adjusted to suit the excitation and emission characteristics of the W11Ni fluorescence probe. Different concentrations of nickel standard solution ranging from 5 nM to 50 μM were gradually added to the test system, and the fluorescence spectrometer was used to observe and record the changes in fluorescence intensity. According to the relationship between fluorescence intensity and nickel ion concentration, a calibration curve was plotted for the quantitative analysis of nickel ion concentration in unknown samples.

[0039] After the system was mixed, a nickel ion stock solution was added. The detection system changed from light yellow to red under 365 nm ultraviolet light, confirming the occurrence of a response. The absorption peak of probe W11Ni at 380 nm shifted to 485 nm after the addition of nickel ions, and the absorption curve is as Figure 2 shown. According to the relationship between fluorescence intensity and nickel ion concentration, a calibration curve was plotted as Figure 3 shown.

[0040] In summary, probe W11Ni was specifically designed to detect nickel ions in e-cigarette aerosols, showing extremely high sensitivity and selectivity. In a specific experiment, we combined the fluorescence probe with a non-standard e-cigarette sample that might contain nickel ions, and the fluorescence intensity increased significantly, showing a significant color change (such as Figure 4 changing from light yellow to red), which was easy to observe and record. This change was further quantitatively analyzed by a fluorescence spectrometer to obtain a fluorescence intensity of 1056.3408 a.u., and the nickel ion concentration was calculated to be 1.223 μM by substituting into the standard curve, successfully detecting the non-standard e-cigarette sample. In particular, the detection sensitivity of this probe for nickel ions is very high, with a linear range from 5 nM to 50 μM and a linear regression coefficient reaching 0.9996, indicating a very high consistency and reliability of its response.

[0041] In addition, the ability of probe W11Ni to rapidly detect nickel ions in e-cigarette aerosols is particularly important for meeting industry safety standards. This probe can rapidly identify and quantify nickel ions in complex samples without complex sample processing or expensive detection equipment. This is particularly important for e-cigarette manufacturers as they can perform rapid and accurate quality control at a relatively low cost to ensure that their products meet national and international safety standards.

[0042] In summary, the probe W11Ni not only achieves efficient and accurate detection of nickel ions in e-cigarette aerosols technically, but also provides a cost-effective solution, offering strong technical support for product safety in the e-cigarette industry. The development of this probe demonstrates its broad application potential in the fields of environmental monitoring and public health, and is expected to promote the development of e-cigarette products towards a safer and more transparent direction.

Claims

1. A small molecule fluorescent probe, characterized in that: The molecular formula of the small molecule fluorescent probe is C 35 H 29 ClN4O6, its structure is shown below: 。 2. The method for preparing the small molecule fluorescent probe according to claim 1, characterized in that The steps include: Step 1: First, add (3,5,5-trimethylcyclohex-2-enylidene)malononitrile, p-hydroxybenzaldehyde, piperidine and anhydrous ethanol into a reactor, heat to 85°C for reaction, and monitor the progress of the reaction by thin layer chromatography; after the reaction is completed, evaporate the solvent to obtain a crude product, and separate by column chromatography to obtain compound I; Step 2: Dissolve the compound i obtained in step 1 and hexamethylenetetramine in trifluoroacetic acid and reflux at 85°C for reaction; after the reaction is completed, pour the reactant into ice water and add hydrochloric acid, filter under reduced pressure, wash the dried solid with cold water, and separate by column chromatography to obtain compound ii; Step 3: Compound ii is dissolved in a mixed solvent of ethanol and dichloromethane, 2-amino-5-chlorobenzamide is added at room temperature, the mixture is heated to 95°C and refluxed for 30 minutes, p-toluenesulfonic acid monohydrate is added, and the reflux reaction is continued for 2 hours; the red suspension after the reaction is cooled to room temperature, DDQ is added in batches, stirred at room temperature for 8-12 hours, the precipitate is filtered and washed, and dried to obtain compound iii; Step 4: dissolve compound iii in dry tetrahydrofuran, add diisopropylethylamine, cool in an ice bath, slowly add allyl formic acid chloride, stir and react at room temperature for 8-12 hours, and obtain the target product after separation and purification; The synthetic route is as follows: 。 3. Use of the small molecule fluorescent probe according to claim 1 in the preparation of nickel ion detection reagents.

4. The use according to claim 3, characterized in that: When you 2+ When the concentration is in the range of 5nM-50µM, the fluorescence intensity of the detection reagent at 485nm is similar to that of Ni 2+ The concentration showed a linear relationship.

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