A method for rapid in situ live detection of nicotine in tobacco leaves

Abstract

The present invention discloses a method for rapid in situ live detection of nicotine in tobacco leaves, comprising the following steps: (1) preparing a series of mixed solutions of NIC and quantitative PA with a concentration gradient; (2) activating the screen-printed electrode; (3) measuring the SWV response thereof, and converting ΔI NIC / ΔI PA Correlate with NIC concentration and draw a standard curve; 4) SWV detects ΔI in living tobacco leaf samples NIC / ΔI PA , and then the NIC content in fresh living tobacco leaves is calculated according to the standard curve. The dual-signal ratiometric electrochemical testing method of the present invention has the advantages of simple operation, easy control, mild reaction conditions, low cost, high sensitivity and fast response speed, and is of great significance for accurately detecting the NIC content in living tobacco leaves.

Description

Technical Field

[0001] The invention belongs to the technical field of tobacco, and in particular relates to a method for rapid in-situ live detection of nicotine (NIC) in tobacco leaves. Background Art

[0002] Nicotine (NIC) is an alkaloid present in tobacco leaves and is a key chemical component of tobacco quality. However, smokers and second-hand smokers suffer greatly from it. Smokers become addicted, and inhaling too much NIC can seriously affect the central nervous system, increase the incidence of lung cancer and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. At present, although it has become common knowledge that smoking is harmful to health, there are still more than 1.2 billion people in the world who consume different tobacco products and become addicted to NIC, resulting in about 4.9 million deaths each year. The content of NIC is a key factor in judging the quality of cigarette products. Therefore, the rapid and accurate determination of the NIC content in tobacco leaves is of great significance to medicine, toxicology and the tobacco industry.

[0003] In recent years, the methods for analyzing and detecting NIC have been continuously developed. In addition to traditional chemical methods and titration methods, electrochemical methods, atomic absorption spectroscopy, gas chromatography, spectrophotometry and high performance liquid chromatography have all been used for the analysis and determination of NIC; among these technologies, electrochemical-based methods are more portable, fast in detection speed and low in cost, and are favored by many researchers; in particular, the emergence of portable electrochemical workstations allows them to be used in conjunction with mobile phones. Compared with traditional electrochemical workstations, they are not only more portable and low in cost, but also more intelligent and can be used for in-situ detection in the field. Therefore, it is crucial to develop a new, sensitive, fast, simple, cheap and intelligent technology to detect the NIC content in living tobacco leaves.

[0004] The present invention is proposed for this purpose. Summary of the invention

[0005] In view of the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a method for rapid in-situ live detection of NIC in tobacco leaves. NIC and acetaminophen (PA) are directly added to the electrolyte by utilizing their high electrochemical activity without any modification of the electrodes; NIC undergoes oxidation-reduction on the screen-printed electrodes, and a significant electrical signal appears; while PA is used as a reference to produce a stable signal; while the PA concentration is kept constant, as the amount of NIC added increases, the electrical signal caused by NIC gradually increases; the electrical signals of NIC and PA are detected by combining a mobile phone with a portable electrochemical workstation, thereby realizing rapid and highly sensitive detection of the ratio dual electrical signals of NIC in live tobacco leaves.

[0006] The present invention adopts the following technical solutions:

[0007] A method for rapid in situ live detection of nicotine in tobacco leaves comprises the following steps:

[0008] (1) adding a certain amount of acetaminophen (PA) solution and different amounts of nicotine (NIC) solution into a buffer solution, mixing them evenly, and preparing a series of mixed solutions of NIC and quantitative PA with a concentration gradient;

[0009] (2) activating the screen printed electrode (SPCE) and then immersing it in different mixed solutions of step (1);

[0010] (3) Connect the screen-printed electrode treated in step (2) to a portable electrochemical workstation via Bluetooth mode, measure its square wave voltammetry (SWV) response, and calculate ΔI NIC / ΔI PA Correlate with NIC concentration and draw a standard curve;

[0011] (4) Using SWV in a portable electrochemical workstation to detect ΔI in live tobacco leaf samples containing PA and NIC NIC / ΔI PA , and then calculate the NIC content in fresh living tobacco leaves based on the standard curve in step (3).

[0012] Preferably, in step (1), the concentration of the PA solution is 5-20 mM, the concentration of the NIC mother solution is 1-20 mM, and the concentration gradient of the NIC in the prepared mixed solution is 10-800 μM.

[0013] Preferably, the preparation method of the mixed solution in step (1) is: preparing a phosphate buffer solution (PBS) with a concentration of 0.05-0.1M and a pH of 4.0-7.0; adding a certain amount of PA solution to the PBS buffer solution under light-proof conditions and stirring; then adding different amounts of NIC mother solution to the above solution under light-proof conditions, dispersing by magnetic stirring, and preparing a series of mixed solutions of NIC and quantitative PA with a concentration gradient; more preferably, the PBS buffer solution has a concentration of 0.1M and a pH of 7.

[0014] Preferably, the screen-printed electrode activation treatment step in step (2) is: immersing the screen-printed electrode in a saturated Na2CO3 solution for constant potential scanning, then washing it with deionized water for 5 to 6 times, and then immersing the screen-printed electrode in a potassium ferrocyanide / potassium ferrocyanide solution for cyclic voltammetry (CV) scanning, so that the redox peak potential difference is 0 to 200 mV, the scanning voltage is -0.2 to 0.6 V, and the scanning rate is 0 to 0.1 V s -1 ; After scanning, rinse repeatedly with deionized water and blow dry naturally.

[0015] Preferably, the SWV equilibrium time of step (3) is 3 s, the enrichment potential is 0 V, the enrichment time is 120 s, the scanning potential is -0.1 to 1 V, the potential increment is 0.01 V, the amplitude is 0.05 V, the frequency is 5 Hz, and the electrochemical signal ratio ΔI of NIC to PA at different NIC concentrations is calculated. NIC / ΔI PA Draw a working curve.

[0016] Preferably, the concentration of potassium ferrocyanide / potassium ferrocyanide solution is 5 mM.

[0017] The present invention has achieved the following beneficial effects:

[0018] 1. The dual-signal ratiometric electrochemical test method of the present invention has the advantages of simple operation, easy control, mild reaction conditions, low cost, high sensitivity and fast response speed, and can effectively avoid the measurement error caused by the fluctuation of single signal intensity, which is an ideal choice for detecting NIC. The principle of the present invention is to use acetaminophen (PA) as an internal reference probe and quickly detect nicotine in tobacco leaves in situ based on bare screen printed electrodes (SPCE).

[0019] 2. The dual-signal ratiometric electrochemical NIC test method of the present invention and the dual-signal ratiometric electrochemical test method constructed by a mobile phone based on bare screen-printed electrodes and a portable electrochemical workstation are reported for the first time. The present invention combines NIC and PA for the first time to prepare a dual-signal ratiometric electrochemical test method, which can realize rapid and efficient detection of NIC content in living tobacco leaves.

[0020] 3. The test method of the present invention detects NIC by simple activation treatment of bare screen printed electrodes (SPCE), and the detection limit can be as low as 1.72 μM, with high sensitivity, which is of great significance for accurately detecting the NIC content in living tobacco leaves. The method of the present invention can also be used for the detection of NIC content in tobacco products, and can also be used for the quantitative detection of NIC in conventional test reagents or solutions and reaction solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a schematic diagram of the steps of the method for chemically detecting NIC in living tobacco leaves of the present invention.

[0022] Figure 2 SWV diagram of different concentrations of NIC and fixed concentration of PA.

[0023] Figure 3 is the electrochemical signal ratio of NIC to PA ΔI NIC / ΔI PA Linear relationship plot with NIC concentration. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its contents.

[0025] The instruments and reagents used in the examples are as follows: (1) Instrument: PalmSens4 portable electrochemical workstation (Reitemate China Co., Ltd.); (1) Reagents: NIC (Yunnan China Tobacco Industry Co., Ltd.), analytical grade; PA (Shanghai Yuanye Biotechnology Co., Ltd.), analytical grade. All other reagents were of analytical grade, and deionized water was used in the experiments.

[0026] Example: Determination of living tobacco leaves (Yunyan 207), schematic diagram of the method steps for in situ determination of NIC in living tobacco leaves Figure 1 shown.

[0027] (1) Prepare a PBS buffer solution with a concentration of 0.1M and pH = 7, and add 10 μL of a 20 mM PA solution dropwise into the PBS buffer solution under light-proof conditions; also add different amounts of 20 mM NIC dropwise into the above PA PBS solution under light-proof conditions, and stir magnetically to mix and disperse, and prepare concentration gradients of 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 60 μM, 70 μM, 80 μM, 100 μM, 120 μM, 140 μM, 160 μM, 180 μM, 200 μM, 240 μM, 280 μM, 320 μM, 360 μM, 400 μM, 440 μM, 500 μM, 600 μM, 700 μM and 800 μM respectively;

[0028] (2) The SPCE was first immersed in a saturated Na2CO3 solution for constant potential activation treatment. After the smartphone and portable electrochemical workstation were turned on, the chronoamperometry (IT method) was selected and the following parameters were set: equilibrium time of 3 s, applied voltage of 1.2 V, and reaction time of 300 s; after the pretreatment was completed, it was washed with deionized water for 5 to 6 times; the pretreated SPCE was immersed in a 5 mM potassium ferrocyanide / potassium ferrocyanide solution for cyclic voltammetry scanning, and the cyclic voltammetry (CV method) was selected and the following parameters were set: equilibrium time of 3 s, scanning voltage of -0.2 to 0.6 V, and scanning rate of 0.1 V s -1 , the scanning number of circles is 3, so that the redox peak potential difference is 100mV; after the scanning is completed, it is repeatedly washed with deionized water and dried naturally;

[0029] (3) Determine the SWV signals of NIC and PA in the mixed solution. Select SWV and set the following parameters: equilibrium time is 3 s, enrichment potential: 0 V, enrichment time: 120 s, scanning potential is -0.1 to 1 V, potential increment is 0.01 V, amplitude is 0.05 V, frequency is 5 Hz, and the electrodes in step (2) are sequentially immersed in the mixed solutions obtained in step (1) to measure the SWV signals of NIC and PA, such as Figure 2 As shown, the electrochemical signal ratio ΔI of NIC to PA at different NIC concentrations NIC / ΔI PA Draw a standard curve; Figure 3 As shown, in ΔI NIC / ΔI PA When ΔI is less than 0.5, NIC / ΔI PA =0.00786C NIC -0.07152; in ΔI NIC / ΔI PA >0.5, ΔI NIC / ΔI PA =0.01887C NIC -0.80377;

[0030] (4) After local minimally invasive treatment of tobacco leaves, drop 100 μL of PBS buffer solution of PA with a concentration of 10 μM on the surface of the leaves. Clamp the living tobacco leaves (Yunyan 207) and the SPCE in step (2) above with a clean glass slide, fix them horizontally with a bracket, connect them to a portable electrochemical workstation, and connect the mobile phone to the portable electrochemical workstation in Bluetooth mode to form an intelligent electrochemical sensor. Select SWV and set the following parameters: equilibrium time of 3 s, enrichment potential: 0 V, enrichment time: 120 s, scanning potential of -0.1 to 1 V, potential increment of 0.01 V, amplitude of 0.05 V, frequency of 5 Hz, and measure the current of NIC in the living tobacco leaves to be 0.039 μA and the current of PA to be 1.467 μA, ΔI NIC / ΔI PA =0.02658, according to the standard curve drawn in step (3) above, ΔI NIC / ΔI PA =0.02658Substitute Figure 3 Linear equation ΔI NIC / ΔI PA =0.00786C NIC -0.07152, the NIC content in living tobacco leaves was calculated to be 12.481 μM.

[0031] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present invention. It should be pointed out that, for those of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A method for rapid in situ live detection of nicotine in tobacco leaves, characterized in that: The following steps are involved: (1) adding a certain amount of acetaminophen solution and different amounts of nicotine solution into a buffer solution, mixing them evenly, and preparing a series of mixed solutions of acetaminophen with a concentration gradient and a quantitative amount of acetaminophen; (2) activating the screen-printed electrode and then immersing it in different mixed solutions of step (1); (3) Connect the screen-printed electrode treated in step (2) to a portable electrochemical workstation via Bluetooth mode, measure its square wave voltammetry response, and I 尼古丁 / Δ I 对乙酰氨基酚 Correlate with nicotine concentration and draw a standard curve; (4) Detection of Δ in live tobacco leaf samples containing acetaminophen and nicotine using square wave voltammetry in a portable electrochemical workstation I 尼古丁 / Δ I 对乙酰氨基酚 , and then calculate the nicotine content in fresh living tobacco leaves according to the standard curve in step (3).

2. The method according to claim 1, characterized in that: In step (1), the concentration of the acetaminophen solution is 5-20 mM, the concentration of the nicotine stock solution is 1-20 mM, and the gradient of a series of nicotine concentrations in the prepared mixed solution is 10-800 μM.

3. The method according to claim 2, characterized in that The preparation method of the mixed solution in step (1) is as follows: prepare a phosphate buffer solution with a concentration of 0.05-0.1 M and a pH of 4.0-7.0; add a certain amount of acetaminophen solution to the phosphate buffer solution under light-proof conditions, and stir; Then, different amounts of nicotine mother solution were added to the above solution under light-proof conditions, and dispersed by magnetic stirring to prepare a series of mixed solutions of nicotine and quantitative acetaminophen with gradient concentrations.

4. The method according to claim 1, characterized in that: The activation treatment step of the screen-printed electrode in step (2) is as follows: the screen-printed electrode is first immersed in a saturated Na2CO3 solution for constant potential scanning, then washed with deionized water for 5 to 6 times, and then the screen-printed electrode is immersed in a potassium ferrocyanide / potassium ferrocyanide solution for cyclic voltammetry scanning, so that the redox peak potential difference is 0 to 200 mV, the scanning voltage is -0.2 to 0.6 V, and the scanning rate is 0 to 0.1 V s -1 ; After scanning, rinse repeatedly with deionized water and blow dry naturally.

5. The method according to claim 1, characterized in that The square wave voltammetry of step (3) and step (4) has an equilibrium time of 3 s, an accumulation potential of 0 V, an accumulation time of 120 s, a scanning potential of -0.1 to 1 V, a potential increment of 0.01 V, an amplitude of 0.05 V, and a frequency of 5 Hz.

6. The method according to claim 4, characterized in that The concentration of potassium ferrocyanide / potassium ferrocyanide solution is 5 mM.

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