A small molecule fluorescent probe and its application in nickel ion detection

Abstract

This invention discloses a small molecule fluorescent probe and its application in nickel ion detection. The small molecule fluorescent probe is based on a dicyanoisophorone (DCI) and hydroxyphenylquinazolinone (HPQ) coupling derivative, with the molecular formula C0. 35 H 29 ClN4O6, its structure is shown below: Compared with traditional metal ion detection techniques, the small molecule fluorescent probe of this invention not only reacts rapidly but is also easy to operate, requiring no professional technicians or expensive equipment, thus greatly reducing detection costs and improving the efficiency of the detection process.

Description

Technical Field

[0001] This invention belongs to the field of organic chemistry, specifically relating to a small molecule fluorescent probe and its application in nickel ion detection. Background Technology

[0002] With the widespread use of e-cigarettes, regulatory agencies and public health organizations are increasingly concerned about the potential presence of heavy metal ions, such as nickel, in e-cigarette vapors, as they may pose a risk to public health. Existing methods for detecting nickel ions typically involve complex sample processing and costly equipment, which poses a challenge to developing product standards for e-cigarettes and monitoring the safety of e-cigarettes on the market. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a small-molecule fluorescent probe and its application in nickel ion detection. Compared with traditional metal ion detection techniques, this small-molecule fluorescent probe not only reacts rapidly but is also easy to operate, requiring no specialized technicians or expensive equipment, thus significantly reducing detection costs and improving the efficiency of the detection process.

[0004] The small molecule fluorescent probe of this invention is based on a conjugated derivative of dicyanoisophorone (DCI) and hydroxyphenylquinazolinone (HPQ), with the molecular formula C. 35 H 29 ClN4O6 has the following structure:

[0005]

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

[0007] Step 1: First, add (3,5,5-trimethylcyclohexyl-2-enylidene)malonium (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. Monitor the reaction progress by thin-layer chromatography. After the reaction is complete, evaporate the solvent to obtain the crude product. Use pure dichloromethane as the eluent by column chromatography to obtain pure compound i (orange fluorescent solid, yield 50%).

[0008] Step 2: Dissolve the above-obtained (3,5,5-trimethylcyclohexyl-2-enylidene)malonitrile (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 the reaction is complete, pour the reactants 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%).

[0009] Step 3: Compound ii (0.5 g, 1.57 mmol) was dissolved in ethanol (15 mL) and dichloromethane (5 mL) to obtain a yellow solution. 2-amino-5-chlorobenzamide (0.6 g, 3.51 mmol) was added at room temperature, and the mixture was heated to 95 °C and refluxed for 30 minutes. Then, p-toluenesulfonic acid monohydrate (0.02 equivalents) was added, and the mixture was refluxed for another 2 hours. The resulting red suspension was cooled to room temperature, and DDQ (1.01 equivalents) was added in portions. The mixture was stirred overnight at room temperature, the precipitate was filtered, washed three times with absolute ethanol, and twice with dichloromethane. After drying, compound iii (dark red powder, yield 42.5%) was obtained.

[0010] Step 4: Compound iii and allyl carbamate chloride were reacted in dry tetrahydrofuran under alkaline conditions to prepare probe W11Ni. Compound iii was dissolved in dry tetrahydrofuran, and after adding diisopropylethylamine and stirring, the mixture was cooled in an ice bath for 5 minutes. Allyl carbamate chloride was then slowly added. The mixture was stirred overnight at room temperature, the solution was extracted, and the solvent was dried by rotary evaporation under reduced pressure. The crude product was separated by column chromatography using a mixture of petroleum ether and ethyl acetate (volume ratio 4:1) to obtain purified probe W11Ni.

[0011] The synthesis route is shown below:

[0012]

[0013] The application of the small molecule fluorescent probe of this invention in the preparation of nickel ion detection reagents.

[0014] 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 concentrations show a linear relationship.

[0015] The detection reagent can detect Ni in e-cigarette atomized fuel. 2+ Content detection.

[0016] This invention provides a rapid, accurate, and cost-effective detection method using a small-molecule fluorescent probe for highly sensitive and selective detection of nickel ions in e-cigarette atomizers. The probe's design ensures specific identification and measurement of nickel ion concentrations even in complex samples containing other ions, which is crucial for assessing whether e-cigarette products meet safety standards. Furthermore, the use of this fluorescent probe provides e-cigarette manufacturers with a tool to comply with national and international safety standards, helping them monitor and adjust nickel content in real time during product design and quality control.

[0017] The fluorescent probe detection method of this invention is fast, sensitive and efficient, and can be widely used in 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 this invention provides important technical support for the setting and implementation of regulatory standards in the e-cigarette industry, and helps to promote the development of e-cigarette products towards a safer and more transparent direction, thus protecting the health and safety of consumers. Attached Figure Description

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

[0020] Figure 2 These are the original UV absorption spectra of the small molecule probe W11Ni and the UV absorption spectra of the response after the addition of nickel ions.

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

[0022] Figure 4 The color change is detected by a small molecule probe when nickel ions in e-cigarette vapors are detected.

[0023] Figure 5 This is a comparison of data between interfering ions and nickel ions when using small molecule probes to detect multiple interfering ions. Compared to interfering ions, nickel ions exhibit a 20-fold increase in signal response. Detailed Implementation

[0024] Implementation Description: This embodiment describes a method using a material named Ni 2+ Test's fluorescent probe detection system is used for the quantitative analysis of nickel ion concentration in e-cigarette smoke or other samples. This detection system is designed for rapid and accurate detection of nickel ions and is suitable for research and industrial quality control.

[0025] Example 1: Preparation of small molecule probe W11Ni

[0026] 1. First, (3,5,5-trimethylcyclohexyl-2-enylidene)malonium (1.5 g, 8.05 mmol), p-hydroxybenzaldehyde (1.8 g, 14.74 mmol), piperidine (1.0 mL), and anhydrous ethanol were added to a single-necked round-bottom flask equipped with a condenser. The reaction mixture was slowly heated to 85 °C and stirred continuously for 5 hours. The reaction progress was monitored by thin-layer chromatography. After the reaction was completed, the solvent was evaporated to obtain the crude product. The crude product was then obtained by column chromatography using pure dichloromethane as the eluent, yielding pure compound i (orange fluorescent solid, 50% yield).

[0027] 2. The (3,5,5-trimethylcyclohexyl-2-enylidene) malononitrile (0.5 g, 1.72 mmol) and hexamethylenetetramine (0.56 g, 3.99 mmol) obtained above were dissolved in trifluoroacetic acid (5.0 mL) and refluxed at 85 °C. After completion, the reaction mixture was poured into 100 g of ice water and 2.0 mL of hydrochloric acid was added. The mixture was filtered under reduced pressure and the dried solid was washed with cold water. The solid was further purified by column chromatography using pure dichloromethane to give pure compound ii (yellow solid, 80% yield).

[0028] 3. A yellow solution was prepared by dissolving compound ii (0.5 g, 1.57 mmol) in ethanol (15 mL) and dichloromethane (5 mL). 2-Amino-5-chlorobenzamide (0.6 g, 3.51 mmol) was added at room temperature. The mixture was heated to 95 °C and refluxed for 30 minutes, followed by the addition of p-toluenesulfonic acid monohydrate (0.02 equivalents), and reflux was continued for 2 hours. The resulting red suspension was cooled to room temperature, and DDQ (1.01 equivalents) was added in portions, with stirring overnight at room temperature. The precipitate was filtered, washed three times with absolute ethanol and twice with dichloromethane, and dried to give compound iii (a dark red powder, yield 42.5%).

[0029] 4. Compound iii (100 mg, 0.213 mmol) and allyl carbamate chloride (272.3 mg, 2.556 mmol) were reacted in 10 mL of dry tetrahydrofuran under alkaline conditions to prepare probe W11Ni. Compound iii was dissolved in dry tetrahydrofuran, and diisopropylethylamine (82.6 mg, 0.639 mmol) was added. After stirring, the mixture was cooled in an ice bath for 5 minutes, and allyl carbamate chloride was slowly added. The mixture was stirred overnight at room temperature, and the solution was extracted and the solvent was dried by rotary evaporation under reduced pressure. The crude product was separated by column chromatography using a mixture of petroleum ether and ethyl acetate (v / v 4:1) to obtain purified probe W11Ni (yellow powder, yield 58.5%).

[0030] Example 2: Fluorescence detection

[0031] The nickel ion fluorescent probe detection method provided by this invention uses a pre-configured Ni 2+ The Test system enables efficient quantitative analysis of nickel ion concentration in analytes. In practice, the analyte is first added to a detection solution containing a W11Ni fluorescent probe, triphenylphosphine (PPh3), and sodium borohydride (NaBH4), according to a pre-configured system. The solution ratio is optimized to ensure specific binding of nickel ions to the fluorescent probe and the resulting change in fluorescence signal.

[0032] After sample preparation, the reaction system was analyzed using a fluorescence spectrometer. The fluorescence spectrometer monitors changes in fluorescence signals in the test solution in real time based on the characteristic excitation and emission wavelengths of the probe. When nickel ions bind to the probe, the fluorescence intensity changes, reflecting the concentration of nickel ions in the sample.

[0033] To achieve quantitative analysis, this invention utilizes a pre-constructed concentration standard curve. The standard curve consists of a nickel standard solution of known concentration and corresponding fluorescence intensity data points. By comparing the relationship between the sample fluorescence intensity and the standard curve, the concentration of nickel ions 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 was fully considered. Through multiple calibrations and verifications, the high accuracy and reliability of the measurement results were ensured. The fluorescence probe W11Ni achieved a response sensitivity of 5 nM (LOD) to nickel ions.

[0034] Example 3: Using Ni 2+ Test system fluorescent probe detection system for detecting nickel ions

[0035] Configure a testing system:

[0036] Solvent and reagent preparation: First, prepare 50 mL of a methanol:water mixture in a ratio of 8:2. Mix methanol and water according to the volume ratio and stir thoroughly to ensure homogeneity. Prepare a 10 mM stock solution of triphenylphosphine (PPh3) in methanol. Dissolve an appropriate amount of triphenylphosphine in methanol and store in a sealed container. Prepare a 10 mM stock solution of sodium borohydride (NaBH4) in water, similarly. Dissolve an appropriate amount of sodium borohydride in water and store in a sealed container.

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

[0038] Before testing, take appropriate amounts from each stock solution and mix them into a 1 mL volume to ensure the working concentration of the W11Ni probe is 20 μM. Simultaneously, ensure the final concentrations of PPh3 and NaBH4 in the test system are both 100 μM. Place the prepared test system in a suitable fluorescence spectrometer and adjust the spectrometer settings to suit the excitation and emission characteristics of the W11Ni fluorescent probe. Gradually add different concentrations of nickel standard solution to the test system, ranging from 5 nM to 50 μM, and observe and record the changes in fluorescence intensity using a fluorescence spectrometer. Based on the relationship between fluorescence intensity and nickel ion concentration, plot a calibration curve for quantitative analysis of nickel ion concentration in unknown samples.

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

[0040] In summary, the W11Ni probe is specifically designed for detecting nickel ions in e-cigarette atomizers, exhibiting extremely high sensitivity and selectivity. In specific experiments, we bound the fluorescent probe to non-standard e-cigarette samples that may contain nickel ions; the fluorescence intensity significantly increased, showing a marked color change (e.g., ...). Figure 4 The change from pale yellow to red was easy to observe and record. Further quantitative analysis using fluorescence spectroscopy yielded a fluorescence intensity of 1056.3408 au. Substituting this into the standard curve, the calculated nickel ion concentration was 1.223 µM, successfully detecting non-standard e-cigarette samples. In particular, the probe exhibited very high sensitivity for nickel ion detection, with a linear range from 5 nM to 50 μM and a linear regression coefficient of 0.9996, indicating highly consistent and reliable response.

[0041] Furthermore, the W11Ni probe's ability to rapidly detect nickel ions in e-cigarette atomizers is particularly important for meeting industry safety standards. This probe can quickly identify and quantify nickel ions in complex samples without requiring sophisticated sample handling or expensive detection equipment. This is especially important for e-cigarette manufacturers, as it allows them to perform rapid and accurate quality control at a lower cost, ensuring their products comply with national and international safety standards.

[0042] In summary, the W11Ni probe not only achieves efficient and accurate detection of nickel ions in e-cigarette atomizers, 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 environmental monitoring and public health, and is expected to drive the development of e-cigarette products towards greater safety and transparency.

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 has the following structure: 。 2. The method for preparing the small molecule fluorescent probe according to claim 1, characterized in that... Includes the following steps: Step 1: First, add (3,5,5-trimethylcyclohexyl-2-enylidene)malonitrile, p-hydroxybenzaldehyde, piperidine and anhydrous ethanol to the reactor, heat to 85°C and react, and monitor the reaction progress by thin-layer chromatography; after the reaction is completed, evaporate the solvent to obtain the crude product, and separate compound i by column chromatography. Step 2: Dissolve compound i obtained in step 1 and hexamethylenetetramine in trifluoroacetic acid and reflux at 85°C; after the reaction is complete, pour the reactants into ice water and add hydrochloric acid, filter under reduced pressure and wash the dried solid with cold water, and separate by column chromatography to obtain compound ii; Step 3: Dissolve compound ii in a mixed solvent of ethanol and dichloromethane, add 2-amino-5-chlorobenzamide at room temperature, heat to 95°C and reflux for 30 minutes, then add p-toluenesulfonic acid and continue reflux for 2 hours; cool the resulting red suspension to room temperature, add DDQ in portions, stir at room temperature for 8-12 hours, filter the precipitate and wash, and dry to obtain compound iii; Step 4: Dissolve compound iii in dry tetrahydrofuran, add diisopropylethylamine, cool in an ice bath, slowly add allyl carboxylic acid chloride, stir the reaction at room temperature for 8-12 hours, and obtain the target product after separation and purification. The synthesis route is shown below: 。 3. The application of the small molecule fluorescent probe according to claim 1 in the preparation of nickel ion detection reagents.

4. The application 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 concentrations show a linear relationship.

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