An electrochemiluminescence sensor and its preparation method, and a method for detecting nicotine

Abstract

The present invention relates to an electrochemiluminescence sensor, a preparation method thereof, and a method for detecting nicotine, belonging to the field of chemical detection technology. The electrochemiluminescence sensor of the present invention includes an electrode and a composite fixed to the electrode, wherein the composite is mainly composed of a metal-organic framework material and an active substance, wherein the active substance is prepared by an amidation reaction between a carboxyl group in tris(4,4-dicarboxylbipyridyl)ruthenium chloride and an amino group in polyethyleneimine. The electrochemiluminescence sensor of the present invention can be used to quantitatively detect nicotine in a solution. When used for quantitative detection of nicotine in a solution, it has the advantages of short detection time and simple operation, as well as high detection accuracy, good sensitivity, and selectivity.

Description

Technical Field

[0001] The present invention relates to an electrochemiluminescence sensor and a preparation method thereof, and a nicotine detection method, belonging to the technical field of chemical detection. Background Art

[0002] Nicotine, also known as (S) 3-[1-methylpyrrolidin-2-yl] pyridine, is a colorless to pale yellow liquid in its pure form with a relative molecular mass of 162.23 g / mol. At the same time, nicotine is an N-cholinergic receptor agonist that can activate cholinergic receptors in the nervous system, such as the adrenal glands, muscles, heart, and brain, thereby producing physiological effects similar to those of acetylcholine. More neuroscience research reports indicate that small doses of nicotine acting on the central nervous system can trigger energy flow, euphoria, and mood elevation. However, long-term accumulation of nicotine can lead to neurotoxicity, resulting in varying degrees of memory and sensory decline, and even the risk of cancer or neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In addition, nicotine is considered the main addictive substance in cigarette products and a major obstacle to quitting smoking.

[0003] To date, although various methods based on gas chromatography, mass spectrometry, spectrophotometry, liquid chromatography and surface-enhanced Raman scattering have been developed for the quantitative detection of nicotine in different matrix samples, these methods require skilled experimenters and complex instruments, and take a long time to analyze. Therefore, at this stage, it is urgent to develop a simple, fast and stable method for detecting nicotine. Summary of the Invention

[0004] The object of the present invention is to provide an electrochemiluminescence sensor, which can solve the problems of complex detection instruments and long detection time in the current quantitative detection of nicotine when used to detect nicotine.

[0005] The second object of the present invention is to provide a method for preparing an electrochemiluminescence sensor, which can solve the problem of long detection time in the current detection instruments prepared for quantitative detection of nicotine.

[0006] The third object of the present invention is to provide a method for detecting nicotine, which can solve the problems of complex detection instruments and long detection time in the current quantitative detection of nicotine.

[0007] In order to achieve the above-mentioned purpose, the technical solution adopted by the electrochemiluminescence sensor of the present invention is:

[0008] An electrochemiluminescence sensor comprises an electrode and a composite fixed on the electrode, wherein the composite is mainly composed of a metal organic framework material and an active substance, wherein the active substance is prepared by an amidation reaction between the carboxyl group in tris(4,4-dicarboxybipyridyl)ruthenium chloride and the amino group in polyethyleneimine.

[0009] The electrochemiluminescence sensor of the present invention can be used to quantitatively detect nicotine in a solution. It has the advantages of short detection time and simple operation, as well as high detection accuracy, good sensitivity, and selectivity. The metal-organic framework in the composite increases the loading capacity of the electrochemiluminescent material and enhances luminescence efficiency, while the active substance acts as a luminescent material, providing an electrochemiluminescent signal.

[0010] Preferably, the structure of the polyethyleneimine is as shown in Formula 1:

[0011]

[0012] In Formula 1, n is an integer greater than 0.

[0013] Preferably, the weight-average molecular weight of the polyethyleneimine is 90,000 to 100,000. If the weight-average molecular weight of the polyethyleneimine is too large, the shape of the synthesized active material will be uneven. If the weight-average molecular weight of the polyethyleneimine is too small, the particle size of the synthesized active material will be too small, the loading amount of the luminescent material will be reduced, and the electrochemiluminescence signal will be weakened.

[0014] Preferably, the mass ratio of tris(4,4-dicarboxybipyridyl)ruthenium chloride to polyethyleneimine is (9-10):10. For example, the mass ratio of tris(4,4-dicarboxybipyridyl)ruthenium chloride to polyethyleneimine is 9:10. Excessive use of tris(4,4-dicarboxybipyridyl)ruthenium chloride can result in uneven morphology of the synthesized active material and composite; insufficient use can result in low luminous efficiency of the active material.

[0015] Preferably, the amidation reaction method comprises the following steps: first activating tris(4,4-dicarboxybipyridyl)ruthenium chloride with a carboxyl activator to obtain an activated intermediate, and then reacting the activated intermediate with polyethyleneimine for not less than 2 hours.

[0016] Preferably, the amidation reaction method comprises the following steps:

[0017] (1) mixing tris(4,4-dicarboxybipyridyl)ruthenium chloride and a carboxyl group activating agent in water to activate the carboxyl group in tris(4,4-dicarboxybipyridyl)ruthenium chloride;

[0018] (2) The system mixed in step (1) is mixed with polyethyleneimine for reaction and purification.

[0019] Preferably, the carboxyl activating agent includes 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide.

[0020] Preferably, the mass ratio of tris(4,4-dicarboxybipyridyl)ruthenium chloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide is (9-9.5):(28.8-30):(5.8-6). For example, the mass ratio of tris(4,4-dicarboxybipyridyl)ruthenium chloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and N-hydroxysuccinimide is 9:28.8:5.8.

[0021] Preferably, in step (1), the mixing time is not less than 15 minutes.

[0022] In order to improve the mixing rate and mixing effect and save reaction time, preferably, in step (2), the polyethyleneimine is used in the form of a polyethyleneimine solution.

[0023] Preferably, the mass fraction of the polyethyleneimine solution is 1-2%. For example, the mass fraction of the polyethyleneimine solution is 1%.

[0024] Preferably, in step (2), the mixing reaction time is not less than 2 hours.

[0025] In order to remove unreacted free tris(4,4-dicarboxybipyridyl)ruthenium chloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide and polyethyleneimine, preferably, in step (2), the purification method includes the following steps: dialyzing the system after the mixed reaction with a dialysis membrane to remove substances with a molecular weight of less than 2000Da in the system; the molecular weight cut-off of the dialysis membrane is not less than 2000Da. It is understandable that since the molecular weight of the product obtained by the amidation reaction is greater than the molecular weight of each raw material, the system after the mixed reaction is dialyzed with a dialysis membrane to remove substances with a molecular weight of less than 2000Da in the system.

[0026] Preferably, the metal organic framework material is prepared by self-assembling a water-soluble zirconium salt and 1,2,4-benzenetricarboxylic acid in water and then removing impurities.

[0027] Preferably, the water-soluble zirconium salt is a water-soluble tetravalent zirconium salt. Preferably, the water-soluble tetravalent zirconium salt is a zirconium halide. For example, the water-soluble tetravalent zirconium salt is zirconium chloride.

[0028] Preferably, the molar ratio of the water-soluble zirconium salt to 1,2,4-benzenetricarboxylic acid is (0.9-1): 1. For example, the molar ratio of the water-soluble zirconium salt to 1,2,4-benzenetricarboxylic acid is 1:1.

[0029] Preferably, the temperature of the self-assembly is 95-110° C. and the time is 40-50 hours. For example, the temperature of the self-assembly is 100° C. and the time is 48 hours.

[0030] Preferably, the mass of water used for every 1.40 g of water-soluble zirconium salt is 20 to 30 g. For example, the mass of water used for every 1.40 g of water-soluble zirconium salt is 20 g.

[0031] Preferably, the self-assembly is performed by allowing an aqueous solution containing a water-soluble zirconium salt and 1,2,4-benzenetricarboxylic acid to stand.

[0032] Preferably, the impurity removal method comprises the following steps: performing solid-liquid separation on the self-assembled system, and then washing and drying the product obtained by the solid-liquid separation.

[0033] Preferably, the washing step is to sequentially wash the product obtained by solid-liquid separation with water and methanol. Preferably, the drying temperature is 70-75°C.

[0034] Preferably, the composite is prepared by a method comprising the following steps: mixing the metal organic framework material and the active substance in water, and performing solid-liquid separation to obtain the composite.

[0035] Preferably, the mass ratio of the metal-organic framework material to the active substance is 1:(0.9-1.1). For example, the mass ratio of the metal-organic framework material to the active substance is 5.4:5.94. If the mass ratio of the metal-organic framework material to the active substance is too small, the composite synthesis and coating will fail, while if the mass ratio is too large, the ECL efficiency of the composite will be low.

[0036] Preferably, the mixing temperature in the preparation method of the composite is room temperature and the mixing time is not less than 24 hours.

[0037] Preferably, the solid-liquid separation in the preparation method of the composite is performed by centrifugation. Preferably, the centrifugation speed is 7000-8000 rpm and the time is 5-10 minutes. For example, the centrifugation speed is 7000 rpm and the time is 5 minutes.

[0038] The technical solution adopted by the preparation method of the electrochemiluminescence sensor of the present invention is:

[0039] A method for preparing the electrochemiluminescence sensor as described above comprises the following steps: coating an aqueous dispersion of the complex onto an electrode, and incubating the electrode to obtain the electrochemiluminescence sensor.

[0040] The preparation method of the electrochemiluminescence sensor of the present invention is simple to operate. When the prepared electrochemiluminescence sensor is used for quantitative detection of nicotine in a solution, it has the advantages of short detection time and simple operation, and has high detection accuracy, good sensitivity and selectivity.

[0041] Preferably, the concentration of the aqueous dispersion of the complex is 0.26-0.3 g / mL. Too high a concentration of the aqueous dispersion of the complex results in excessive signal intensity (overrange), while too low a concentration of the aqueous dispersion of the complex results in too low a signal intensity.

[0042] Preferably, the coating density of the aqueous dispersion of the composite on the electrode is 20 to 30 μL / 100 mm 2 For example, the coating density of the aqueous dispersion of the composite on the electrode is 25 μL / 100 mm 2 .

[0043] Preferably, the incubation temperature is 30-40° C. and the time is 9-14 h. For example, the incubation temperature is 37° C. and the time is 12 h.

[0044] Preferably, the electrode is an indium tin oxide electrode. Preferably, the surface to be coated of the electrode contains hydroxyl groups.

[0045] To impart hydroxyl groups to the surface of the indium tin oxide electrode to be coated, the surface of the indium tin oxide electrode is preferably subjected to a hydroxylation treatment. Preferably, the hydroxylation treatment involves soaking the indium tin oxide electrode in an alkaline solution. Preferably, the alkaline solution is a sodium hydroxide solution. Preferably, the concentration of the sodium hydroxide solution is 1 to 2 mol / L. Preferably, the soaking time is no less than 1 hour.

[0046] The technical solution adopted by the nicotine detection method of the present invention is:

[0047] A method for detecting nicotine comprises using the electrochemiluminescence sensor as described above to detect nicotine in a solution to be tested, and determining the nicotine concentration in the solution to be tested based on the electrochemiluminescence signal obtained by the detection and a standard curve.

[0048] The nicotine detection method of the present invention has the advantages of short detection time and simple operation, and has high detection accuracy, good sensitivity and selectivity. The detection limit of the nicotine detection method of the present invention is as low as 1.9×10 -12 mol / L.

[0049] Preferably, when detecting nicotine in the test solution, the voltage scanning range is 0 to -1.5V, and the voltage of the photomultiplier tube is 500 to 600V.

[0050] It is understandable that the solution to be tested contains an electrolyte, which is persulfate.

[0051] Preferably, the standard curve is ΔECL=-1330.138Lg CNicotine +943.136, where ΔECL represents the change in electrochemiluminescence signal intensity, which is equal to the difference between the electrochemiluminescence signal intensity measured when there is no nicotine in the electrolyte and the electrochemiluminescence signal intensity measured when there is nicotine in the electrolyte, C Nicotine Represents the concentration of nicotine. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 The electrochemiluminescence spectra obtained by testing in electrolytes containing different concentrations of nicotine in Example 3;

[0053] Figure 2 Schematic diagram of the relationship between the difference in electrochemiluminescence signal intensity detected in electrolytes containing different nicotine concentrations and the nicotine concentration and the linear equation obtained by fitting in Example 3;

[0054] Figure 3 Schematic diagram of the results obtained when the bare ITO electrode was used as an electrochemiluminescence sensor in Experimental Example 1 and the electrochemiluminescence sensors prepared in Example 2 and Comparative Example 1 were tested for electrochemiluminescence signals (the nicotine concentration in the electrolyte used in the test was 0);

[0055] Figure 4 In Experimental Example 1, the bare ITO electrode was used as an electrochemiluminescence sensor. At the same time, the electrochemiluminescence sensors prepared in Example 2 and Comparative Example 1 were tested for electrochemiluminescence signals (the nicotine concentration in the electrolyte used in the test was 5.0×10 -4 mol / L) obtained;

[0056] Figure 5 Schematic diagram of the test results of the quenching effect of nicotine on the electrochemiluminescence sensors prepared in Comparative Example 1 and Example 2 in Experimental Example 1;

[0057] Figure 6 Schematic diagram of the principle of the electrochemiluminescence sensor based on quenching reaction in Experimental Example 2;

[0058] Figure 7 TEM images of the metal-organic framework material (Zr-MOF) and the composite (Ru-BPEI@Zr-MOF) in Experimental Example 3;

[0059] Figure 8UV-vis absorption spectra of polyethyleneimine, tris(4,4-dicarboxybipyridyl)ruthenium chloride, and the active material (Ru-BPEI) in Experimental Example 3, Fourier transform infrared (FTIR) spectra, a schematic diagram of the Zeta potential test results of the metal-organic framework material (Zr-MOF) and the active material (Ru-BPEI), and a schematic diagram of the cyclic voltammetry experimental results of the bare ITO electrode and the electrochemiluminescence sensor prepared in Example 2;

[0060] Figure 9 Schematic diagram of the results of optimizing the experimental parameters in Experimental Example 4;

[0061] Figure 10 Schematic diagram of the test results of the electrochemiluminescence sensor on different matrix samples in Experimental Example 5;

[0062] Figure 11 This is the electrochemiluminescence spectrum obtained from the nicotine test in the actual sample in Experimental Example 6. DETAILED DESCRIPTION

[0063] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0064] All nicotine in the examples and experimental examples of the present invention was obtained from the Zhengzhou Tobacco Research Institute in China, ZrCl4, tris(4,4'-dicarboxylic acid-2,2'-bipyridyl)ruthenium(II) dichloride, also known as tris(4,4-dicarboxylbipyridyl)ruthenium chloride ([Ru(dcbpy)3 2+ ]Cl2), polyethyleneimine (BPEI), 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and 1,2,4-benzenetricarboxylic acid were purchased from MacLean Reagent Co., Ltd. (Shanghai, China). L-Cysteine (L-Cys), glucose (Glu), glycerol (Gly), glutamic acid (GA), and propylene glycol (1,2-Propanediol, PG) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China). The weight-average molecular weight of polyethyleneimine (BPEI) was 100,000, and its chemical formula is shown below:

[0065]

[0066] 1. Specific embodiments of the electrochemiluminescence sensor of the present invention are as follows:

[0067] Example 1

[0068] The electrochemiluminescence sensor of this embodiment includes an electrode and a composite fixed on the electrode. The electrode is an indium tin oxide electrode with a hydroxyl group on its surface. The composite is composed of a metal-organic framework material and an active substance. The active substance is prepared by an amidation reaction between the carboxyl group in tris(4,4-dicarboxybipyridyl)ruthenium chloride and the amino group in polyethyleneimine.

[0069] 2. Specific examples of the preparation method of the electrochemiluminescence sensor of the present invention are as follows:

[0070] Example 2

[0071] The preparation method of the electrochemiluminescence sensor of this embodiment is the same as the preparation method of the electrochemiluminescence sensor of Example 1, and specifically comprises the following steps:

[0072] (1) Preparation of metal-organic framework materials

[0073] 1.40 g of zirconium tetrachloride (ZrCl4) was dissolved in 10 mL of deionized water and ultrasonically treated for 10 minutes to obtain a zirconium chloride solution; then 1.26 g of 1,2,4-benzenetricarboxylic acid was dispersed in 10 mL of deionized water and stirred for 10 minutes to obtain a 1,2,4-benzenetricarboxylic acid solution; the zirconium chloride solution and the 1,2,4-benzenetricarboxylic acid solution were mixed and stirred for 20 minutes to obtain a mixed solution, and then the mixed solution was sealed in a PTFE (polytetrafluoroethylene)-lined autoclave, and the autoclave was transferred to a preheated oven at 100°C and allowed to stand for 48 hours. After cooling to room temperature, the mixture in the autoclave was centrifuged, and the solid obtained by centrifugation was washed with water and methanol respectively, and the number of water and methanol washings was 3 times. Finally, the washed solid was dried at 70°C to obtain a metal organic framework material (represented by Zr-MOF);

[0074] (2) Preparation of active substances

[0075] 9 mg of tris(4,4-dicarboxybipyridyl)ruthenium chloride was dissolved in 2 mL of ultrapure water and ultrasonically treated for 5 min to obtain a tris(4,4-dicarboxybipyridyl)ruthenium chloride solution. Then, 28.8 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and 5.8 mg of N-hydroxysuccinimide (NHS) were added to the tris(4,4-dicarboxybipyridyl)ruthenium chloride solution, and magnetic stirring was used to mix for 15 min to activate the carboxyl group to obtain a mixed solution containing an activated intermediate. Then, 1 g of a 1% polyethyleneimine solution was added to the mixed solution containing the activated intermediate and stirred for 2 h. Polyethyleneimine reacts with the activated intermediate, and then the reaction system is dialyzed (dialysis time is 60 hours) using a dialysis membrane with a molecular weight cutoff of 2000Da to purify the system, removing substances with a molecular weight less than 2000Da from the system to obtain a retentate, which is a dispersion containing the active substance. The active substance in the retentate is detected by a chemical analysis instrument, and the results show that the concentration of the active substance is 5.94mg / mL. The experimental results also indicate that unreacted polyethyleneimine or tris(4,4-dicarboxybipyridyl)ruthenium chloride has no effect on the test performance of the prepared electrochemiluminescent sensor. The prepared active substance is stored at 4°C for future use.

[0076] (3) Preparation of complex

[0077] 5.4 mg of the metal organic framework material prepared in step (1) was dispersed in 1 mL of deionized water to obtain a metal organic framework material dispersion, and then 1 mL of the dispersion containing the active substance prepared in step (2) (at a concentration of 5.94 mg / mL) was added to the metal organic framework material dispersion, and stirred for 24 h to obtain a dispersion containing a complex, and then the dispersion containing the complex was centrifuged (at a speed of 7000 rpm for 5 min). The solid obtained by centrifugation was the complex, and then the complex was dispersed in 5 mL of deionized water to obtain a complex dispersion with a concentration of 0.26 g / mL, and the complex dispersion was stored at 4°C for later use;

[0078] (4) Preparation of electrochemiluminescence sensor

[0079] An indium tin oxide (ITO) electrode sheet with a length of 100 mm, a width of 100 mm, and a thickness of 1.1 mm was first ultrasonically cleaned 3 times with ethanol, and then ultrasonically cleaned 3 times with water. The cleaned indium tin oxide (ITO) electrode sheet was then dried and then immersed in a NaOH solution with a concentration of 1 mol / L at room temperature for 1 h. The immersed indium tin oxide electrode sheet was then washed and dried in sequence to obtain an indium tin oxide electrode sheet containing hydroxyl groups on the surface. Then 25 μL of the composite dispersion prepared in step (3) was dropped onto a surface (area of 100 mm×100 mm) of the indium tin oxide electrode sheet containing hydroxyl groups on the surface, and incubated at 37°C for 12 h. At this time, the composite was completely adsorbed on the indium tin oxide electrode sheet to obtain an electrochemiluminescence sensor.

[0080] Comparative Example 1

[0081] The preparation method of the electrochemiluminescence sensor of this comparative example specifically comprises the following steps:

[0082] (1) A dispersion containing the active substance (concentration: 5.94 mg / mL) was prepared according to step (2) of Example 1;

[0083] (2) An indium tin oxide (ITO) electrode sheet with a length of 100 mm, a width of 100 mm, and a thickness of 1.1 mm was first ultrasonically cleaned 3 times with ethanol, and then ultrasonically cleaned 3 times with water. The cleaned indium tin oxide (ITO) electrode sheet was then dried and then immersed in a NaOH solution with a concentration of 1 mol / L for 1 h. The immersed indium tin oxide electrode sheet was then washed and dried in sequence to obtain an indium tin oxide electrode sheet containing hydroxyl groups on the surface. Then, 25 μL of the dispersion containing active substance (concentration of 5.94 mg / mL) prepared in step (1) was dropped onto one surface (area of 100 mm×100 mm) of the indium tin oxide electrode sheet containing hydroxyl groups on the surface, and incubated at 37°C for 12 h. At this time, the active substance was completely adsorbed on the indium tin oxide electrode sheet to obtain an electrochemiluminescence sensor.

[0084] 3. Specific examples of the nicotine detection method of the present invention are as follows:

[0085] Example 3

[0086] The nicotine detection method of this embodiment specifically comprises the following steps:

[0087] (1) Establish a standard curve

[0088] The electrochemiluminescence sensor prepared in Example 2 was tested for electrochemiluminescence signal using an MPI-E electrochemiluminescence analyzer. The electrolyte used in the test was a phosphate buffer solution containing nicotine and potassium persulfate. The voltage scanning range was 0 to -1.5 V, and the photomultiplier tube (PMT, Program Map The voltage of the electrochemical luminescence sensor (Table) was set to 500 V, the electrochemiluminescence sensor prepared in Example 2 was used as the working electrode, the platinum wire was used as the counter electrode, and the Ag / AgCl (saturated KCl) was used as the reference electrode; the electrolyte was prepared by mixing nicotine, potassium persulfate, and a phosphate buffer solution, and the phosphate buffer solution was prepared by mixing NaCl, NaH2PO4, KCl, KH2PO4, and water, and the concentrations of NaCl, NaH2PO4, KCl, and KH2PO4 were 0.137, 0.01, 0.0027, and 0.002 mol / L, respectively; the pH of the phosphate buffer solution was 7.4, the concentration of potassium persulfate in the electrolyte was 0.1 mol / L, and the concentration of nicotine in the electrolyte was 1.0×10 -12 mol / L~5.0×10 -4 mol / L; By testing electrolytes containing different concentrations of nicotine, the relationship between nicotine concentration and electrochemiluminescence signal was established, which can be expressed by a linear equation: ΔECL = -1330.138Lg CNicotine +943.136(R 2 =0.9953), where ΔECL represents the change in electrochemiluminescence signal intensity (ΔECL, ΔECL=ECL0-ECL, ECL and ECL0 represent the electrochemiluminescence signal intensity when nicotine is present in the electrolyte and the electrochemiluminescence signal intensity when nicotine is not present in the electrolyte, respectively), C Nicotine represents the concentration of nicotine, and the linear range of the linear equation is 1.0×10 -11 ~5.0×10 -4 mol / L, the detection limit was 1.9×10 -12 mol / L; The electrochemiluminescence spectra obtained by testing in electrolytes containing different concentrations of nicotine are shown in the figure below. Figure 1 As shown in FIG, the difference between the electrochemiluminescence signal intensity detected in the electrolyte without nicotine and the electrochemiluminescence signal intensity detected in the electrolyte containing different concentrations of nicotine (ΔECL, ΔECL=ECL0-ECL, ECL and ECL0 represent the electrochemiluminescence signal intensity when nicotine is present in the electrolyte and the electrochemiluminescence signal intensity when nicotine is not present in the electrolyte, respectively) is plotted against the nicotine concentration, and the linear equation obtained by fitting is plotted, as shown in FIG. Figure 2 As shown;

[0089] (2) Detecting the nicotine concentration in the test solution

[0090] A tobacco leaf peeled from a Huangshan cigarette was immersed in 10 mL of distilled water for 12 hours, coarsely filtered, and the filtrate was centrifuged at 13,000 rpm for 10 minutes to remove large particulate matter. Then, 1 mL of the supernatant in the filtrate was diluted 25 times with a phosphate buffer solution containing potassium persulfate to obtain a test solution; the phosphate buffer solution containing potassium persulfate was prepared by mixing potassium persulfate and a phosphate buffer solution, and the phosphate buffer solution was prepared by mixing NaCl, NaH2PO4, KCl, KH2PO4 and water, and the concentrations of NaCl, NaH2PO4, KCl, and KH2PO4 were 0.137, 0.01, 0.0027, and 0.002 mol / L, respectively. The pH of the phosphate buffer solution was 7.4, and the concentration of potassium persulfate in the phosphate buffer solution containing potassium persulfate was 0.1 mol / L.

[0091] Then, the solution to be tested was used as the electrolyte, and the electrochemiluminescence signal test was performed according to the method in step (1). The electrochemiluminescence signal (ΔECL) obtained by the test was substituted into the standard curve in step (1) to calculate the nicotine concentration in the solution to be tested, and the result was 1.69×10 -6 mol / L.

[0092] Experimental Example 1

[0093] In order to evaluate the performance of the bare ITO electrode, the electrochemiluminescence sensors prepared in Example 2 and Comparative Example 1, the bare ITO electrode was used as an electrochemiluminescence sensor. At the same time, the electrochemiluminescence sensors prepared in Example 2 and Comparative Example 1 were subjected to electrochemiluminescence signal testing according to the method in step (1) of Example 3. The nicotine concentration in the electrolyte used in the test was 0 mmol / L. The results are shown in FIG. Figure 3 As shown. Figure 2 It can be seen that the bare ITO electrode shows a very weak signal, the signal obtained by the electrochemiluminescence sensor prepared in Comparative Example 1 is significantly enhanced, and the signal obtained by the electrochemiluminescence sensor prepared in Example 2 is further enhanced, confirming that the metal organic framework material prepared in Example 2 has an excellent loading capacity for active substances. When nicotine is present (the nicotine concentration in the electrolyte is 5.0×10 -4 mol / L), the signals detected by the electrochemiluminescence sensors prepared in Comparative Example 1 and Example 2 were somewhat weakened ( Figure 4 shown).

[0094] The quenching effect of nicotine on the electrochemiluminescence sensors prepared in Comparative Example 1 and Example 2 can be evaluated based on the difference in electrochemiluminescence signal intensities. The difference in electrochemiluminescence signal intensities (ΔECL) obtained when the electrochemiluminescence sensors prepared in Comparative Example 1 and Example 2 are tested without nicotine and with nicotine is plotted. The results are shown in FIG. Figure 5 As shown, ΔECL = ECL0 - ECL, where ECL and ECL0 represent the electrochemiluminescence signal intensity in the presence and absence of nicotine in the electrolyte, respectively. The results show that nicotine has a superior quenching effect on the electrochemiluminescence sensor prepared in Example 2, indicating that the electrochemiluminescence sensor prepared in Example 2 can be used for nicotine analysis.

[0095] Experimental Example 2

[0096] Based on Experimental Example 1, a simple analysis of the principle of the electrochemiluminescence sensor based on quenching reaction was conducted, such as Figure 6 As shown. Figure 6 It can be seen that when the electrochemiluminescent sensor prepared in Example 2 comes into contact with a solution containing nicotine, the divalent ruthenium in the complex (Ru(Ⅱ)-BPEI@Zr-MOF) fixed on the electrode is converted into a monovalent ruthenium complex (Ru(Ⅰ)-BPEI@Zr-MOF), and the persulfate (S2O8 2- ) is reduced to sulfate radical anion (SO4 ·- ), nicotine (C 10 H 14 The pyrrole group in N2 is replaced by sulfate radical anion (SO4 ·- ) oxidation, competitive oxidation occurs, resulting in the monovalent ruthenium complex (Ru(I)-BPEI@Zr-MOF) and the strong oxidant SO4 ·- The reaction of converting to the free radical complex (Ru(II)*-BPEI@Zr-MOF) weakens, whereby the excited state returns to the ground state and the accompanying luminescence phenomenon weakens. Figure 6 The nicotinic acid in it represents nicotinic acid, and products represents carbon-containing compounds.

[0097] Experimental Example 3

[0098] In order to characterize the structure of the metal organic framework material (Zr-MOF) and the composite (Ru-BPEI@Zr-MOF) prepared in Example 2, the morphology of the metal organic framework material (Zr-MOF) and the composite (Ru-BPEI@Zr-MOF) was characterized by transmission electron microscopy (TEM). The results are as follows: Figure 7 As shown, Figure 7 a is the TEM image of metal organic framework material (Zr-MOF), Figure 7 b is the TEM image of the composite (Ru-BPEI@Zr-MOF). Figure 7It can be seen that the hydrothermally synthesized Zr-MOF has a cubic structure with an average diameter of about 132 nm, and the composite (Ru-BPEI@Zr-MOF) has a circular structure with a higher average diameter of about 232 nm.

[0099] In addition, the UV-vis absorption spectra of polyethyleneimine, tris(4,4-dicarboxybipyridyl)ruthenium chloride and active material (Ru-BPEI) in Example 2 were tested using an ultraviolet-visible light analyzer. The results are as follows: Figure 8 As shown in a, Figure 8 In a, BPEI represents polyethyleneimine, Ru(II) represents tris(4,4-dicarboxybipyridyl)ruthenium chloride, Ru(II)+BPEI represents a mixture of tris(4,4-dicarboxybipyridyl)ruthenium chloride and polyethyleneimine, and Ru-BPEI represents an active substance. Figure 8 As can be seen from the UV-vis curve of polyethyleneimine, there is no obvious absorption peak, while tris(4,4-dicarboxybipyridyl)ruthenium chloride displays two characteristic peaks at 296nm and 465nm, which belong to the ligand-based π→π* transition and metal-ligand charge transfer (MLCT) (dπ(Ru)→π*(dcbpy)). In addition, the UV-vis curve of tris(4,4-dicarboxybipyridyl)ruthenium chloride is identical to the UV-vis curve of a mixture of tris(4,4-dicarboxybipyridyl)ruthenium chloride and polyethyleneimine. However, compared to tris(4,4-dicarboxybipyridyl)ruthenium chloride, the characteristic peaks of the active material (Ru-BPEI) show an offset due to the conjugation effect between the bipyridyl ring of tris(4,4-dicarboxybipyridyl)ruthenium chloride and the -NH2 of polyethyleneimine.

[0100] At the same time, the Fourier transform infrared (FTIR) spectra of polyethyleneimine (BPEI), tris(4,4-dicarboxybipyridyl)ruthenium chloride (Ru(II)) and active material (Ru-BPEI) in Example 2 were measured using a PerkinElmer GX spectrometer (PerkinElmer Co., Waltham, MA). The results are as follows: Figure 8 As shown in b. Figure 8 b It can be seen that for tris(4,4-dicarboxybipyridyl)ruthenium chloride, the stretching vibrations of OH and C=O of -COOH are at 3380 and 1720 cm -1 Compared with tris(4,4-dicarboxybipyridyl)ruthenium chloride, polyethyleneimine and active material (Ru-BPEI) have peaks at 3371, 3266, 2934 and 2830 cm -1 A peak appears at 1598 cm, which is attributed to the stretching vibration of NH and CH in the polyethyleneimine chain. For the active material (Ru-BPEI) sample, a peak appears at 1598 cm -1An amide bond appeared at 1720 cm -1 The -COOH vibration peak at 180 nm disappeared, confirming that the active material (Ru-BPEI) was successfully synthesized from polyethyleneimine and tris(4,4-dicarboxybipyridyl)ruthenium chloride through an amide bond.

[0101] Then, the Zeta potential of the metal organic framework material (Zr-MOF) and the active material (Ru-BPEI) was tested using a Zeta potential analyzer (Zetasizer Nano-ZS system, Malvern, UK). Figure 8 c. Figure 8 c It can be seen that the Zeta potentials of the metal-organic framework material (Zr-MOF) and the active substance (Ru-BPEI) are -38.24 mV and 10.99 mV, respectively, indicating that the metal-organic framework material (Zr-MOF) can adsorb the positively charged active substance (Ru-BPEI) through electrostatic interaction.

[0102] Finally, cyclic voltammetry experiments were conducted on the bare ITO electrode and the electrochemiluminescence sensor prepared in Example 2 (using EC laboratory instruments). The cyclic voltammetry experiments were conducted in a solution containing 0.1 mol / L KCl and 5.0×10 -3 mol / L [Fe(CN)6] 3- / 4- The scanning was carried out in a phosphate buffer solution (0.1 mol / L, pH = 7.4) at a scan rate of 50 mV / s. Figure 8 As shown in d, Figure 8 The ITO in d represents a bare ITO electrode, and Ru-BPEI@Zr-MOF / ITO represents the electrochemiluminescence sensor prepared in Example 2. Figure 8 d shows that the exposed ITO electrode shows [Fe(CN)6] 3- / 4- Compared with the redox peak current corresponding to the bare ITO electrode, the redox peak current corresponding to the electrochemiluminescence sensor prepared in Example 2 is significantly enhanced, indicating that Zr-MOF has good conductivity and can promote electron transfer, and also confirms the successful preparation of the electrochemiluminescence sensor.

[0103] Experimental Example 4

[0104] In order to obtain the best nicotine detection performance, the experimental parameters were optimized. An electrochemiluminescence sensor was prepared according to the preparation method of the electrochemiluminescence sensor in Example 2, except that the mass of the metal organic framework material in step (3) was adjusted so that the mass ratio of the metal organic framework material to the active substance was 0.2:1, 0.5:1, 1:1, 1.5:1, and 2:1, respectively, to obtain the corresponding electrochemiluminescence sensors. The prepared electrochemiluminescence sensor was then subjected to an electrochemiluminescence signal test according to the method in step (1) of Example 3. The nicotine concentrations in the electrolyte used in the test were 0 and 5.0×10 -4 mol / L, the mass ratio of the metal organic framework material and the active substance (abscissa) and the change value of the electrochemiluminescence signal intensity ΔECL (ordinate) obtained by the test were plotted. The results are shown in Figure 2. Figure 9 As shown in a, ΔECL=ECL0-ECL, ECL and ECL0 represent the electrochemiluminescence signal intensity when nicotine is present in the electrolyte and the electrochemiluminescence signal intensity when nicotine is not present in the electrolyte, respectively. Figure 9 It can be seen from a that when the mass ratio of the metal-organic framework material to the active substance is 1:1, the intensity of the electrochemiluminescence signal obtained by the test is the largest.

[0105] In addition, electrochemiluminescence sensors were prepared according to the preparation method of the electrochemiluminescence sensor of Example 2, except that the incubation time in step (4) was adjusted to 2 h, 6 h, 12 h, 16 h, and 24 h, respectively, to obtain corresponding electrochemiluminescence sensors. The prepared electrochemiluminescence sensors were then tested for electrochemiluminescence signals according to the method in step (1) of Example 3. The nicotine concentrations in the electrolyte used in the test were 0 and 5.0 × 10 -4 mol / L, the incubation time (abscissa) and the electrochemiluminescence signal intensity change value ΔECL (ordinate) obtained by the test were plotted. The results are shown in Figure 2. Figure 9 b, wherein ΔECL=ECL0-ECL, ECL and ECL0 represent the electrochemiluminescence signal intensity measured when nicotine is present in the electrolyte and the electrochemiluminescence signal intensity measured when nicotine is not present in the electrolyte, respectively. Figure 9 As shown in Figure 2, ΔECL increases with increasing incubation time, reaching its peak at 12 h. After that, ΔECL begins to decrease continuously. Therefore, 12 h is the optimal incubation time.

[0106] Finally, the electrochemiluminescence sensor prepared in Example 2 was tested for electrochemiluminescence signal according to the method in step (1) of Example 3, except that the pH of the phosphate buffer solution was adjusted to 5, 6, 7, 7.4, and 8, respectively, and the nicotine concentration in the electrolyte used in the test was 0 and 5.0×10-4 mol / L, the pH of the electrolyte (abscissa) and the change value of the electrochemiluminescence signal intensity ΔECL (ordinate) obtained by the test were plotted, and the results are as follows Figure 9 c, where ΔECL=ECL0-ECL, ECL and ECL0 represent the electrochemiluminescence signal intensity when nicotine is present in the electrolyte and the electrochemiluminescence signal intensity when nicotine is not present in the electrolyte, respectively. Figure 9 c It can be seen that as the pH of the phosphate buffer solution gradually increases, the intensity of ΔECL gradually increases and reaches the highest at 7.4, so the optimal pH of the phosphate buffer solution is 7.4.

[0107] Experimental Example 5

[0108] In order to evaluate the matrix effect of the electrochemiluminescence sensor prepared in Example 2, the electrochemiluminescence signal of the electrochemiluminescence sensor prepared in Example 2 was tested according to the method in step (1) of Example 3, except that the nicotine in the electrolyte was replaced by the following matrix samples: L-cysteine (L-cys), glucose (Glu), glycerol (Gly), glutamic acid (GA) and propylene glycol (PG), respectively. The concentrations of L-cysteine (L-cys), glucose (Glu), glycerol (Gly), glutamic acid (GA) and propylene glycol (PG) in the electrolyte were all 0.1 mol / L, and the nicotine concentration in the electrolyte was 5.0×10 - 4 mol / L. Plot the electrochemiluminescence signal intensity change value ΔECL (ordinate) and substance type (abscissa) obtained from the test of each substance, as shown in the figure. Figure 10 As shown, ΔECL=ECL0-ECL, ECL and ECL0 represent the electrochemiluminescence signal intensity when nicotine is present in the electrolyte and the electrochemiluminescence signal intensity when nicotine is not present in the electrolyte, respectively. Figure 10 It can be seen that the electrochemiluminescence sensor prepared in Example 2 is not affected by the matrix when detecting nicotine.

[0109] Experimental Example 6

[0110] In order to evaluate the detection effect of the electrochemiluminescence sensor prepared in Example 2 on nicotine in actual samples, the nicotine content in cigarettes purchased from a supermarket (containing about 0.9 mg of nicotine per cigarette) was tested using step (2) of the method in Example 3 (three samples were tested in parallel). The electrochemiluminescence spectrum obtained from the test is shown in FIG. Figure 11As shown, the difference in electrochemiluminescence signal intensity (ΔECL = ECL0 - ECL, where ECL and ECL0 represent the electrochemiluminescence signal intensity in the presence and absence of nicotine in the electrolyte, respectively) obtained from the test was applied to the standard curve to calculate the nicotine content. The calculated results showed that the nicotine content of one cigarette was 0.688 ± 0.09 mg. The reference nicotine content of one cigarette marked on the cigarette box is ~0.900 mg. Therefore, the nicotine content of the actual sample detected by the electrochemiluminescence sensor prepared in Example 2 is close to the theoretical value, indicating that the method of Example 3 can be used to detect the nicotine content in tobacco samples.

[0111] At the same time, the step (2) of the method in Example 3 was used to test the concentration of 1.0×10 -11 , 1.0×10 -8 , 5.0×10 -4 mol / L nicotine test solution, the actual value, detection value, recovery rate and relative standard deviation of the nicotine concentration in the nicotine test solution are shown in Table 1.

[0112] Table 1 Detection results of different concentrations of nicotine test solutions

[0113] Actual value (mol / L) Detection value (mol / L) Recovery rate (%) Relative standard deviation (%) <![CDATA[1.0×10 -11 ]]> <![CDATA[9.82×10 -12 ]]> 98.20 4.14 <![CDATA[1.0×10 -8 ]]> <![CDATA[9.63×10 -9 ]]> 96.30 2.95 <![CDATA[5.0×10 -4 ]]> <![CDATA[4.95×10 -4 ]]> 99.00 7.27

[0114] As can be seen from Table 1, the difference between the detected value and the theoretical value does not exceed 7.27%, indicating that the method of Example 3 has good detection accuracy.

[0115] In order to evaluate the effect of metal organic framework materials on electrochemiluminescence sensors, electrochemiluminescence sensors were prepared according to the preparation method of the electrochemiluminescence sensor in Example 2, except that zirconium chloride in step (1) was replaced by copper nitrate, zinc nitrate, magnesium nitrate or iron nitrate, or 1,2,4-benzenetricarboxylic acid was replaced by terephthalic acid, 1,3,5-benzenetricarboxylic acid or 2,5-dihydroxy-terephthalic acid. The prepared electrochemiluminescence sensors were then subjected to electrochemiluminescence signal testing according to the method in step (1) of Example 3. The nicotine concentrations in the electrolyte used in the test were 0 mmol / L and 5.0×10 -4mol / L, and then calculated the difference in electrochemiluminescence signal intensity (ΔECL) between each electrochemiluminescence sensor tested without nicotine and with nicotine. The results showed that the difference in electrochemiluminescence signal intensity (ΔECL) obtained for each electrochemiluminescence sensor was significantly smaller than the difference in electrochemiluminescence signal intensity (ΔECL) obtained for the electrochemiluminescence sensor prepared in Example 2 under the same conditions. Calculations indicate that when the structure of the metal-organic framework material is changed, the difference in electrochemiluminescence signal intensity (ΔECL) obtained for each electrochemiluminescence sensor is no greater than 25% of the difference in electrochemiluminescence signal intensity (ΔECL) obtained for the electrochemiluminescence sensor prepared in Example 2 under the same conditions.

Claims

1. A method for detecting nicotine, characterized in that: The following steps are involved: An electrochemiluminescence sensor is used to detect nicotine in a test solution, and the nicotine concentration in the test solution is determined based on the electrochemiluminescence signal obtained by the detection and a standard curve. The electrochemiluminescence sensor includes an electrode and a complex fixed on the electrode. The complex is composed of a metal-organic framework material and an active substance. The active substance is prepared by an amidation reaction between a carboxyl group in tris(4,4-dicarboxybipyridyl)ruthenium chloride and an amino group in polyethyleneimine. The metal-organic framework material is prepared by self-assembly of a water-soluble zirconium salt and 1,2,4-benzenetricarboxylic acid in water and then undergoing impurity removal.

2. The method for detecting nicotine according to claim 1, wherein: The structure of the polyethyleneimine is shown in Formula 1: In Formula 1, n is an integer greater than 0.

3. The method for detecting nicotine according to claim 2, wherein: The weight average molecular weight of the polyethyleneimine is 90,000 to 100,000.

4. The method for detecting nicotine according to claim 1, wherein: The mass ratio of tris(4,4-dicarboxybipyridyl)ruthenium chloride to polyethyleneimine is (9-10):

10.

5. The method for detecting nicotine according to any one of claims 1 to 4, wherein: The amidation reaction method comprises the following steps: firstly activating tris(4,4-dicarboxylbipyridyl)ruthenium chloride with a carboxyl activator to obtain an activated intermediate, and then reacting the activated intermediate with polyethyleneimine for not less than 2 hours.

6. The method for detecting nicotine according to claim 1, wherein: The molar ratio of the water-soluble zirconium salt to 1,2,4-benzenetricarboxylic acid is (0.9-1):1; the temperature of the self-assembly is 95-110° C., and the time is 40-50 hours.

7. The method for detecting nicotine according to claim 1 or 6, wherein: The self-assembly is performed by allowing an aqueous solution containing a water-soluble zirconium salt and 1,2,4-benzenetricarboxylic acid to stand.

8. The method for detecting nicotine according to claim 1, wherein: The composite is prepared by a method comprising the following steps: mixing a metal organic framework material and an active substance in water, and performing solid-liquid separation to obtain the composite; the mass ratio of the metal organic framework material to the active substance is 1:(0.9-1.1).

9. The method for detecting nicotine according to claim 1, wherein: The preparation method of the electrochemiluminescence sensor comprises the following steps: coating the aqueous dispersion of the complex on an electrode, and incubating the electrode to obtain the electrochemiluminescence sensor.

10. The method for detecting nicotine according to claim 9, wherein: The concentration of the aqueous dispersion of the complex is 0.26-0.3 g / mL; the coating density of the aqueous dispersion of the complex on the electrode is 20-30 μL / 100 mm 2 .

11. The method for detecting nicotine according to claim 9 or 10, wherein: The incubation temperature is 30-40° C. and the incubation time is 9-14 hours.

Images