阅读说明：本技术 一种磁固相萃取结合碳点荧光探针检测电子烟中烟碱的方法 (Method for detecting nicotine in electronic cigarette by combining magnetic solid phase extraction with carbon dot fluorescent probe ) 是由 徐雪芹 陈志燕 蒋宏霖 孟冬玲 周芸 贾海江 刘会杰 王学杰 于 2021-09-18 设计创作，主要内容包括：本发明公开了一种磁固相萃取结合碳点荧光探针检测电子烟中烟碱的方法。本发明采用醋酸铜及邻苯二胺为前驱,以乙醇为溶剂,微波法合成铜/氮掺杂荧光碳点(Cu,N-CDs),在270nm激发波长下,Cu,N-CDs在294nm和580nm出现两个发射峰。基于烟碱与Cu,N-CDs荧光内滤效应,烟碱对两个发射峰均产生荧光猝灭,建立高灵敏、选择性强烟碱检测新方法,检出限为0.060μg/g。样品检测中,结合磁固相萃取吸附基体干扰物质。将本方法应用于电子烟中烟碱的检测分析,结果与色谱方法相符。方法具有灵敏度高、特异性强的特点、操作简单、快速。(The invention discloses a method for detecting nicotine in electronic cigarettes by combining magnetic solid-phase extraction with a carbon dot fluorescent probe. Copper acetate and o-phenylenediamine are used as precursors, ethanol is used as a solvent, a microwave method is used for synthesizing copper/nitrogen-doped fluorescent carbon dots (Cu, N-CDs), and under the excitation wavelength of 270nm, two emission peaks of the Cu and N-CDs appear at 294nm and 580 nm. Based on the fluorescent internal filtering effect of nicotine and Cu, N-CDs, the nicotine generates fluorescent quenching on two emission peaks, a novel high-sensitivity and selective strong nicotine detection method is established, and the detection limit is 0.060 mug/g. In the sample detection, the magnetic solid phase extraction is combined to adsorb matrix interfering substances. The method is applied to detection and analysis of nicotine in the electronic cigarette, and the result is consistent with a chromatographic method. The method has the characteristics of high sensitivity and strong specificity, and is simple and rapid to operate.)

1. A method for detecting nicotine in electronic cigarettes by combining magnetic solid phase extraction with carbon dot fluorescent probes is characterized by comprising the following steps:

(1) making a nicotine working curve: adding 50-100 mul of water-soluble copper/nitrogen-doped fluorescent carbon dots and 0-100 mg/L nicotine standard solution into a 10mL colorimetric tube with a plug, diluting the solution to a scale with a pH 6.5-7.0 buffer solution, shaking up, standing for 5-10min, measuring the fluorescence intensity F, F at 294nm by taking 270nm as an excitation wavelength₀The fluorescence intensity of 0 nicotine concentration is plotted on the abscissa as the nicotine concentration, (F)₀-F)/F₀Drawing a standard curve for a vertical coordinate to obtain a regression equation;

(2) sample processing

The electronic cigarette liquid: mixing the electronic cigarette liquid and an isopropanol aqueous solution according to the weight ratio of 1: 100-150, performing vortex for 1-2min, performing centrifugal separation, and fixing the volume of the supernatant to obtain a nicotine extracting solution, wherein the weight volume percentage concentration of the isopropanol aqueous solution is 10-15%;

magnetic solid phase extraction: taking 1mL of nicotine extract in the above steps, adding 5mL of deionized water, and adding Fe₃O₄0.1-0.2mg of magnetic nanoparticles, mixing in a vortex manner for 30-60 seconds, and performing phase separation by an additional magnet, wherein the supernatant is nicotine extract;

(3) and (3) sample determination: adding 50-100 μ L of water-soluble copper/nitrogen-doped fluorescent carbon dots into 10mL colorimetric tube with plug, adding the nicotine extract prepared in step (2), diluting with pH 6.5-7.0 buffer solution to scale, shaking, standing for 5-10min, measuring 294nm fluorescence intensity F with 270nm as excitation wavelength, and calculating (F)₀-F)/F₀And (4) substituting the regression equation in the step (1) to calculate the nicotine content of the sample.

2. The method for detecting nicotine in electronic cigarettes by combining magnetic solid-phase extraction with carbon dot fluorescent probes according to claim 1, wherein the copper/nitrogen-doped fluorescent carbon dots are prepared by the following method: dissolving copper acetate and o-phenylenediamine with the same weight in absolute ethyl alcohol, after complete dissolution, performing microwave digestion for 1h at 300 ℃ and 2000W, cooling to room temperature, centrifuging for 20-25min at 10000r/min, filtering supernate with a 0.22 mu m filter membrane, and performing dialysis treatment for 24-48h by using a dialysis bag with the molecular weight cutoff of 3000-3500Da to obtain copper/nitrogen doped fluorescent carbon dots; wherein the weight volume ratio of the copper acetate, the o-phenylenediamine and the absolute ethyl alcohol is 1: 40-50.

3. The method for detecting nicotine in electronic cigarette by combining magnetic solid phase extraction with carbon dot fluorescent probe according to claim 1, wherein Fe₃O₄The magnetic nano-particles are prepared by the following method: firstly, 1 part of FeCl₂·4H₂O and 2.5 parts FeCl₃·6H₂Dispersing O in 10-15 parts of deionized water; then at 80 ℃ N₂Adding 2 parts of 2mol/LNaOH under protection; in order to make the material uniform in particle size, 0.05 part of sodium dodecyl sulfate is added and stirred for 0.5 h; and separating the prepared product by using a magnet, washing the product for 3-5 times by using deionized water, and finally drying the product in a vacuum oven at the temperature of 60 ℃.

4. The method for detecting nicotine in electronic cigarettes by combining magnetic solid-phase extraction and carbon dot fluorescent probes according to any one of claims 1 to 3, wherein the pH 6.5-7.0 buffer solution is Tris-HCl buffer solution.

Technical Field

The invention relates to the technical field of chemical analysis and detection, in particular to a method for detecting nicotine in electronic cigarettes by combining magnetic solid-phase extraction with a carbon dot fluorescent probe.

Background

Nicotine, commonly known as nicotine, is the main addictive component of tobacco, is the most important alkaloid in tobacco, and accounts for about 1.5 percent of the mass of tobacco and is almost 95 percent of the total alkaloid content in cigarettes. The nicotine salt has stable property and strong penetrating power, and can meet the requirement of people on nicotine in a short time. Nicotine can be dissolved in water and alcohol rapidly, and can be absorbed by human body easily through mouth, nose and bronchus mucosa. Nicotine adhering to the skin surface can "seep" into the body. The death causing amount of nicotine in the cigarette to human bodies is 50-70 mg, which is equivalent to the content of nicotine in 20-25 cigarettes. If the nicotine of one cigar or three cigarettes is injected into the vein of a human body at one time, the human body can die in 3-5 minutes. Has effect in killing insects. The detection of nicotine is important. The national tobacco industry standard YC/T468-2013 describes that the total plant alkaloid of tobacco and tobacco products is measured by a continuous flow (potassium thiocyanate) method.

Carbon Dots (CDs) are a nano material with good biocompatibility, low toxicity or no toxicity, and are widely applied in the research fields of chemical analysis, biological imaging, photocatalysis and the like. The research group previously studied the resonance energy transfer between carbon dots and metal quantum dots, established a fluorescence measurement method of nicotine based on the fluorescence of metal quantum dots enhanced by nicotine, and applied for invention patent (201810207247.9).

Disclosure of Invention

The invention aims to provide a method for detecting nicotine in electronic cigarettes by combining magnetic solid-phase extraction with a carbon dot fluorescent probe, which is characterized in that a new nicotine fluorescence detection method is established by utilizing selective fluorescence quenching of copper/nitrogen doped fluorescent carbon dots by nicotine. Copper acetate and o-phenylenediamine are used as precursors, ethanol is used as a solvent, a microwave method is used for synthesizing copper/nitrogen-doped fluorescent carbon dots (Cu, N-CDs), and under the excitation wavelength of 270nm, two emission peaks of the Cu and N-CDs appear at 294nm and 580 nm. Based on the fluorescent internal filtering effect of nicotine and Cu, N-CDs, the nicotine generates fluorescent quenching on two emission peaks, a novel high-sensitivity and selective strong nicotine detection method is established, and the detection limit is 0.060 mug/g. In the sample detection, the magnetic solid phase extraction is combined to adsorb matrix interfering substances. The method is applied to detection and analysis of nicotine in cigarettes and electronic cigarettes, and the result accords with a chromatographic method. The method has the characteristics of high sensitivity, strong specificity, simple and rapid operation and the like.

A method for detecting nicotine by combining magnetic solid phase extraction with carbon dot fluorescent probes comprises the following steps:

(1) making a nicotine working curve: adding 50-100 mul of water-soluble copper/nitrogen-doped fluorescent carbon dots and 0-100 mg/L nicotine standard solution into a 10mL colorimetric tube with a plug, diluting the solution to a scale with a pH 6.5-7.0 buffer solution, shaking up, standing for 5-10min, measuring the fluorescence intensity F, F at 294nm by taking 270nm as an excitation wavelength₀The fluorescence intensity of 0 nicotine concentration is plotted on the abscissa as the nicotine concentration, (F)₀-F)/F₀And drawing a standard curve for a vertical coordinate to obtain a regression equation.

(2) Sample processing

The electronic cigarette liquid: mixing the electronic cigarette liquid and an isopropanol aqueous solution according to the weight ratio of 1: 100-150, whirling for 1-2min, centrifuging, and diluting the supernatant to a constant volume to obtain the nicotine extract, wherein the weight volume percentage concentration of the isopropanol aqueous solution is 10-15%.

Magnetic solid phase extraction: taking 1mL of nicotine extract in the above steps, adding 5mL of deionized water, and adding Fe₃O₄0.1-0.2mg of magnetic nano particles, vortex mixing for 30-60 seconds, and carrying out phase separation by an additional magnet, wherein the supernatant is nicotine extraction liquid.

(3) And (3) sample determination: adding 50-100 μ L of water-soluble copper/nitrogen-doped fluorescent carbon dots into 10mL colorimetric tube with plug, adding the nicotine extract prepared in step (2), diluting with pH 6.5-7.0 buffer solution to scale, shaking, standing for 5-10min, measuring 294nm fluorescence intensity F with 270nm as excitation wavelength, and calculating (F)₀-F)/F₀And (4) substituting the regression equation in the step (1) to calculate the nicotine content of the sample.

Further, the copper/nitrogen doped fluorescent carbon dot is prepared by the following method: dissolving copper acetate and o-phenylenediamine with the same weight in absolute ethyl alcohol, after complete dissolution, performing microwave digestion for 1h at 300 ℃ and 2000W, cooling to room temperature, centrifuging for 20-25min at 10000r/min, filtering supernate with a 0.22 mu m filter membrane, and performing dialysis treatment for 24-48h by using a dialysis bag with the molecular weight cutoff of 3000-3500Da to obtain copper/nitrogen doped fluorescent carbon dots; wherein the weight volume ratio of the copper acetate, the o-phenylenediamine and the absolute ethyl alcohol is 1: 40-50.

Further, said Fe₃O₄The magnetic nano-particles are prepared by the following method: firstly, 1 part of FeCl₂·4H₂O and 2.5 parts FeCl₃·6H₂O is dispersed in 10-15 parts of deionized water. Then at 80 ℃ N₂2 parts of 2mol/LNaOH are added under protection. To homogenize the particle size of the material, 0.05 part of sodium lauryl sulfate is subsequently added and stirred for 0.5 h. And separating the prepared product by using a magnet, washing the product for 3-5 times by using deionized water, and finally drying the product in a vacuum oven at the temperature of 60 ℃.

Further, the pH 6.5-7.0 buffer solution is Tris-HCl buffer solution.

The invention has the advantages that:

1. the invention utilizes the fluorescent internal filtering effect between the nicotine and the nitrogen-copper doped fluorescent carbon dots, the nicotine generates fluorescent quenching on the emission peak, and a new method for detecting the high-sensitivity and high-selectivity nicotine is established.

2. The synthesis method of the copper-nitrogen doped carbon dots used in the invention is simple, the fluorescence yield is high, under the excitation wavelength of 270nm, the copper-nitrogen doped carbon dots have two emission peaks at 294nm and 580nm, and nicotine has fluorescence quenching effect on the two emission peaks, and the two emission peaks are in a linear relation in a certain concentration range, and can be measured by adopting corresponding emission wavelengths according to different interference conditions of actual samples.

3. In the measurement of the sample, Fe is used₃O₄Magnetic nanoparticle matrix dryingThe elimination of interference plays a good role in purification and improves the detection accuracy.

Drawings

FIG. 1 shows dual-emission fluorescence quenching of Cu, N-CDs by nicotine in example 1, for detection by nicotine fluorescence probe.

Fig. 2, 3 and 4 show the effect of coexisting ions and sugars (glucose, maltose and sucrose), amino acids (glycine, cysteine, methionine and glutamine), organic acids (oxalic acid, citric acid, succinic acid, malic acid, gallic acid, phloroglucinol acid) and phenolic substances on nicotine.

Detailed Description

The technical solutions of the present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.

FIG. 1 shows dual-emission fluorescence quenching of Cu, N-CDs by nicotine in example 1, for detection by nicotine fluorescence probe.

Fig. 2, 3 and 4 show the effect of coexisting ions and saccharides (glucose, maltose and sucrose), amino acids (glycine, cysteine, methionine and glutamine), organic acids (oxalic acid, citric acid, succinic acid, malic acid, gallic acid, phloroglucinol acid) and phenolic substances on nicotine, and it can be seen from the figure that nicotine has a good selectivity for simulated fluorescence quenching of Cu, N-CDs.

Example 1:

preparation of carbon quantum dots

1. Preparing a copper/nitrogen doped fluorescent carbon dot: dissolving 0.5g of copper acetate and 0.5g of o-phenylenediamine in 20mL of absolute ethanol, digesting for 1h in a microwave digestion instrument at the temperature of 300 ℃ and the temperature of 2000W after completely dissolving, cooling to room temperature, centrifuging for 20-25min at 10000r/min, filtering a supernatant with a 0.22 mu m filter membrane, and then dialyzing for 24-48h by using a dialysis bag with the molecular weight cutoff of 3000-3500Da to obtain the copper/nitrogen doped fluorescent carbon dots;

2、Fe₃O₄preparing magnetic nanoparticles: first 3.5g FeCl₂·4H₂O and 8.75g FeCl₃·6H₂O was dispersed in 35mL of deionized water. Then at the temperature of 80 ℃, the temperature of the mixture is controlled,N₂7.5mL NaOH (2mol/L) was added with protection. To homogenize the particle size of the material, 0.175g of sodium lauryl sulfate was subsequently added and stirred for 0.5 h. And separating the prepared product by using a magnet, washing the product for 3-5 times by using deionized water, and finally drying the product in a vacuum oven at the temperature of 60 ℃.

3. Making a nicotine working curve: adding 50-100 μ L water soluble copper/nitrogen doped fluorescent carbon spot into 10mL colorimetric tube with plug, adding 0.5mL nicotine standard solution of 0, 1, 20, 40, 60, 80, 100mg/L, diluting with pH 6.5-7.0 buffer solution to scale, shaking, standing for 5-10min, measuring 294nm fluorescence intensity F, F with 270nm as excitation wavelength₀The fluorescence intensity of 0 nicotine concentration is plotted on the abscissa as the nicotine concentration, (F)₀-F)/F₀For the ordinate, a standard curve is drawn, see fig. 1, and the regression equation, correlation coefficient, relative standard deviation, linear range, etc. obtained are shown in table 1.

4. Method specificity investigation: FIG. 2 and FIG. 3 show nicotine concentrations of 20mg/L, K⁺、Na⁺、Mg²⁺、Ca²⁺、Zn²⁺、Mn²⁺、Co^{2 +}、Ni²⁺、Pb²⁺、Fe²⁺、Fe³⁺、Al³⁺(ii) a The method comprises the following steps of adding copper/nitrogen-doped fluorescent carbon dots into saccharides (glucose, maltose and sucrose), amino acids (glycine, cysteine, methionine and glutamine), organic acids (oxalic acid, citric acid, succinic acid, malic acid, gallic acid and phloroglucinol acid) and phenolic substances respectively, wherein the concentration of the interference substances is 200mg/L, only nicotine has an obvious quenching effect, and other substances hardly have the quenching effect, so that the method has good selection specificity.

5. Nicotine content determination of electronic cigarette smoke liquid

(1) Mixing the electronic cigarette liquid and an isopropanol aqueous solution according to the weight ratio of 1: 100-150, whirling for 1-2min, centrifuging, and diluting the supernatant to a constant volume to obtain the nicotine extract, wherein the weight volume percentage concentration of the isopropanol aqueous solution is 10-15%.

(2) E, processing the tobacco juice sample of the electronic cigarette: weighing 0.1g of electronic cigarette liquid sample, accurately measuring to 0.0001g, adding 10mL of 15% isopropanol aqueous solution by volume, mixing for 1min by vortex, centrifuging, collecting the centrifuged isopropanol aqueous solution supernatant, and placing in a 250mL volumetric flask with deionized water to obtain nicotine extract.

(3) Magnetic solid-phase extraction: taking 1mL of nicotine extract in the above steps, adding 5mL of deionized water, and adding Fe₃O₄0.1-0.2mg of magnetic nano particles, vortex mixing for 30-60 seconds, and carrying out phase separation by an additional magnet, wherein the supernatant is nicotine extraction liquid.

(4) And (3) nicotine determination: adding 50-100 μ L of water-soluble copper/nitrogen-doped fluorescent carbon dots into 10mL colorimetric tube with plug, adding the nicotine extract prepared in step (1), diluting with pH 6.5-7.0 buffer solution to scale, shaking, standing for 5-10min, measuring 294nm fluorescence intensity F with 270nm as excitation wavelength, and calculating (F)₀-F)/F₀And substituting the regression equation in the step 3 to calculate the nicotine content of the sample to be 15.3 mg/g.

TABLE 1 Linear equation, correlation coefficient, relative standard deviation, Linear Range

Example 2: determination of nicotine in tobacco juice of fruit-type tobacco tar electronic cigarette

1. Preparing a copper/nitrogen doped fluorescent carbon dot: the same as example 1;

2、Fe₃O₄preparing magnetic nanoparticles: the same as example 1;

3. making a nicotine working curve: the same as example 1;

4. method specificity investigation: the same as example 1;

5. measuring the nicotine content of the electronic cigarette liquid, namely mixing the electronic cigarette liquid and an isopropanol aqueous solution according to the weight ratio of 1: 100-150, whirling for 1-2min, centrifuging, and diluting the supernatant to a constant volume to obtain the nicotine extract, wherein the weight volume percentage concentration of the isopropanol aqueous solution is 10-15%.

6. Nicotine content determination of electronic cigarette smoke liquid

Mixing the electronic cigarette liquid and an isopropanol aqueous solution according to the weight ratio of 1: 100-150, whirling for 1-2min, centrifuging, and diluting the supernatant to a constant volume to obtain the nicotine extract, wherein the weight volume percentage concentration of the isopropanol aqueous solution is 10-15%.

(1) E, processing the tobacco juice sample of the electronic cigarette: weighing 0.1g of electronic cigarette liquid sample, accurately measuring to 0.0001g, adding 10mL of 15% isopropanol aqueous solution by volume, mixing for 1min by vortex, centrifuging, collecting the centrifuged isopropanol aqueous solution supernatant, and placing in a 250mL volumetric flask with deionized water to obtain nicotine extract.

(2) Magnetic solid-phase extraction: taking 1mL of nicotine extract in the above steps, adding 5mL of deionized water, and adding Fe₃O₄0.1-0.2mg of magnetic nano particles, vortex mixing for 30-60 seconds, and carrying out phase separation by an additional magnet, wherein the supernatant is nicotine extraction liquid.

(3) And (3) nicotine determination: adding 50-100 mu L of water-soluble copper/nitrogen-doped fluorescent carbon dots into a 10mL colorimetric tube with a plug, adding the nicotine extraction liquid prepared in the step (1), diluting the nicotine extraction liquid to a scale by using a buffer solution with pH 6.5-7.0, shaking uniformly, standing for 5-10min, measuring the fluorescence intensity F of 294nm by using 270nm as an excitation wavelength, calculating (F0-F)/F0, substituting the regression equation in the step 3, and calculating the nicotine content of the sample to be 11.6 mg/g.

Example 3: determination of nicotine in tobacco extract type tobacco tar electronic cigarette

1. Preparing a copper/nitrogen doped fluorescent carbon dot: the same as example 1;

2、Fe₃O₄preparing magnetic nanoparticles: the same as example 1;

3. making a nicotine working curve: the same as example 1;

4. method specificity investigation: the same as example 1;

5. determination of nicotine content in samples: as in example 1, the measurement result was 10.2 mg/g.

Example 4: determination of nicotine in tobacco liquid of electronic cigarette with Chinese herbal medicine tobacco tar

1. Preparing a copper/nitrogen doped fluorescent carbon dot: the same as example 1;

2、Fe₃O₄preparing magnetic nanoparticles: the same as example 1;

3. making a nicotine working curve: the same as example 1;

4. method specificity investigation: the same as example 1;

5. determining the nicotine content in the tobacco juice of the electronic cigarette: as in example 1, the measurement result was 18.1 mg/g.

The results of the standard recovery rate and relative standard deviation tests of the samples 1-4 are shown in Table 2, and the results are compared with the method for measuring nicotine in tobacco industry standard YC/T468-2013 tobacco and tobacco products.

Table 2 recovery with standard addition and relative standard deviation (n ═ 5)

From the results of table 2, it can be seen that: compared with the YC/T246-2008 determination method, the method for detecting nicotine by using the magnetic solid phase extraction combined with the carbon dot fluorescent probe has the same result, but has the advantages of less processing steps, short used time, low processing cost, simple and convenient operation, no need of large-scale instruments and equipment, high recovery rate and relative standard deviation of 2.1-3.6 percent, and good accuracy and precision.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.