CN108398411B - A method of nicotine is detected based on fluorescence resonance energy transfer - Google Patents
A method of nicotine is detected based on fluorescence resonance energy transfer Download PDFInfo
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Abstract
The invention discloses a kind of methods based on fluorescence resonance energy transfer detection nicotine, carbon quantum dot with better optical property is applied to fluorescence resonance energy transfer with metal quantum point, using carbon quantum dot as energy donor, using metal quantum point as energy acceptor, construct novel energy transfer system, nicotine can be such that the fluorescence in system enhances, and establish the high sensitivity of nicotine and the detection method of selectivity；This method takes full advantage of the better optical property and difference of fluorescent carbon quantum dot Yu fluorescence metal quantum dot, establishes nicotine high sensitivity, strong and easy to operate, the quick new detecting method of selectivity.
Description
Technical field
The present invention relates to a kind of methods based on fluorescence resonance energy transfer detection nicotine, belong to detection technique field.
Background technique
Nicotine also known as nicotine belong to pyridine race alkaloid, are the endemic elements in tobacco；With medical in recent years, cigarette
The rapid development of the numerous areas such as careless industry, agricultural, chemical industry, it is growing day by day to the requirement of natural nicotine in the market；Quickly, simple
Just, the content for accurately measuring nicotine in nicotine sample and nicotine product is necessary to enterprise's production.Existing nicotine analysis
Detection method have gravimetry, analysis by titration, polarimetry, IR spectrum quantitative analysis method, atomic absorption spectrophotometry,
Red, orange, green, blue, yellow (ROGBY), potentiometry, polarogarphy etc., and these methods cut both ways.Fluorescence resonance energy transfer refers to
In different fluorophors, if the absorption light of the fluorescence emission spectrum of a fluorophor (donor) and another group (receptor)
Spectrum have certain overlapping, when the distance between two groups is suitable (generally less than 50 nm), that is, can be observed fluorescent energy by
The phenomenon that donor is shifted to receptor, i.e. donor fluorescent quenching and acceptor fluorescence enhancing.Carbon quantum dot is that a kind of partial size is generally less than
The new carbon of 10nm.It has excellent optical property, it is adjustable excitation and transmitting behavior, higher fluorescent stability,
The features such as lower toxicity and good biocompatibility；Metal quantum point is a kind of semiconductor nanocrystal particle, has width
Excitation spectrum and narrow emission spectrum, the features such as good light stability, chemical stability is strong；Quantum dot is dividing as fluorescence probe
The application for analysing chemical field becomes hot spot, has no report relevant to technical solution of the present invention.
Summary of the invention
The purpose of the present invention is to provide a kind of measuring methods of the short nicotine of easy to operate, high sensitivity, detection time；
Fluorescence resonance energy transfer occurs using between carbon quantum dot and metal quantum point, carbon quantum dot is as energy donor, metal quantum
Point is used as energy acceptor, constructs novel energy transfer system, and nicotine can be such that metal quantum point fluorescence in system enhances,
Under mild acid conditions, the fluorescence value added of metal quantum point and the concentration of nicotine are in good linear relationship in a certain range,
To establish the new method of measurement trace nicotine.
The present invention is based on the methods of fluorescence resonance energy transfer detection nicotine, comprising the following steps:
(1) 20~40 μ L water-soluble metal amounts the production of nicotine working curve: are sequentially added in 10mL color-comparison tube
Son point solution, 60~80 μ L Water-soluble carbon quantum dot solution and 0.5 mL nicotine standard solution, are buffered molten with pH 6.0~6.5
Liquid is diluted to scale, shakes up, and stands 8~10min, and using 300nm as excitation wavelength, excitation and transmite slit are 5nm, measurement
The fluorescence intensity F of 600nm, calculates (F-F0)/F0Regression equation, F indicates that fluorescence of metal quantum point in the presence of nicotine is strong
Degree, F0Respectively indicate fluorescence intensity of metal quantum point in the presence of no nicotine；
(2) sample treatment
Cigarette sample: accurately weighing cigarette cut tobacco 1.000g and be placed in flask, and 80~120 mL dehydrated alcohols, leaching is added
Bubble 12~for 24 hours, separates filtrate, adds 80~120 mL soaked in absolute ethyl alcohol 12~for 24 hours, filtering, merging filtrate, constant volume in
In 250 mL volumetric flasks, nicotine extracting solution is obtained；
Tobacco juice for electronic smoke:, will be electric in the ratio of 1.000g tobacco juice for electronic smoke addition 100~150mL isopropanol water solution
Sub- cigarette tobacco juice is dissolved in isopropanol water solution, and the concentration of volume percent of isopropanol water solution is 5~15%, and vortex mixed 1~
3min, centrifuge separation, and take the clear liquid after centrifugation as nicotine extracting solution；
Tobacco sample: accurately weighing dry tobacco leaf powder 1.000g and be placed in a beaker, and 0.5~1.0 mol/L is added
30~50 mL of HCl solution, slightly boiled 10~15min, filters while hot in small fire, reusable heat water washing residue 3~4 times, filtrate
Merge, 0.5~1.0 mol/L NaOH solution tune pH to 7 is added, is settled in 250mL volumetric flask with water and obtains nicotine extracting solution；
(3) sample measures: 20~40 μ L water-soluble metal quantum dot solutions, 60 are sequentially added in 10mL color-comparison tube
~80 μ L Water-soluble carbon quantum dot solution and the nicotine extracting solution of 5mL step (2) preparation are dilute with 6.0~6.5 buffer solution of pH
It releases to scale, shakes up, stand 8~10min, using 300nm as excitation wavelength, excitation and transmite slit are 5nm, measure 600nm
Fluorescence intensity F, calculate (F-F0)/F0, step (1) regression equation is substituted into, sample nicotine content is calculated.
The Water-soluble carbon quantum dot solution the preparation method comprises the following steps: weigh 10.0g polyethyleneimine liquid, be added to 90mL
In ultrapure water, 5~8min of ultrasound mixes well it, and is transferred to ptfe autoclave, in 180~200 DEG C heat 5~
7h is first 0.22 μm of membrane filtration with aperture after cooled to room temperature, is then 3000~3500Da with molecular cut off
Bag filter carry out dialysis treatment for 24 hours, obtain Water-soluble carbon quantum dot solution.
The water-soluble metal quantum dot solution the preparation method comprises the following steps: by 50~70 mL, 0.01 mol/L CdCl2It is molten
Liquid, 250~300 μ L thioacetic acid are added in three-necked flask, under magnetic agitation after mixing, are slowly added to 1.0 mol/L
NaOH adjusts pH to 11, in N2After 30~45min of the lower stirring of protection removes the oxygen in mixed liquor, it is added dropwise 0.1~0.2
mol/L Na2S•9H2O aqueous solution 5mL continues in N2Protection is lower to stir 4h, and water-soluble CdS metal quantum point solution can be obtained.
The buffer solution that 6.0~6.5 buffer solution of pH is made of sodium dihydrogen phosphate and disodium hydrogen phosphate.
The centrifuge separation is 5~10min of centrifugation at 3000~6000r/ of centrifugation rate min.
The present invention has the advantages that
1, by with better optical property carbon quantum dot and metal quantum point be applied to fluorescence resonance energy transfer, with carbon
Quantum dot is as energy donor, using metal quantum point as energy acceptor, constructs novel energy transfer system, nicotine can make
Fluorescence in system enhances, and establishes the high sensitivity of nicotine and the detection method of selectivity；
2, method has the characteristics that good stability, high sensitivity, selectivity are good；
3, method is easy to operate, quick, does not use organic solvent, environmentally protective.
Detailed description of the invention
Fig. 1 is the Absorption and emission spectra of carbon quantum dot and metal quantum point, and wherein a is the absorption spectrum of carbon quantum dot, b
For the absorption spectrum of metal quantum point, c is the emission spectrum of carbon quantum dot, the emission spectrum of d metal quantum point；
Fig. 2 is the concentration of fixed metal quantum point, changes the spectrogram of carbon quantum dot concentration；
Fig. 3 is the concentration of fixed carbon quantum dot, changes the spectrogram of metal quantum point concentration；
Fig. 4 is enhancing result schematic diagram of the different nicotinic densities to metal quantum point fluorescence intensity.
Specific embodiment
The present invention is further described below with reference to embodiment, but the scope of protection of the present invention is not limited thereto.
Embodiment 1: the measurement of nicotine in cigarette sample
1, the preparation of Water-soluble carbon quantum dot solution: weighing 10.0g polyethyleneimine liquid and be added in 90mL ultrapure water,
Ultrasonic 5min mixes well it, and is transferred to ptfe autoclave, after 200 DEG C of heating 5h, cooled to room temperature,
Be first 0.22 μm of membrane filtration with aperture, after with the bag filter that molecular cut off is 3500Da carry out dialysis treatment for 24 hours, obtain
Water-soluble carbon quantum dot solution；
2, the preparation of water-soluble metal quantum dot: by 0.01 mol/L CdCl of 50mL2Solution, 250 μ L thioacetic acid add
Enter in three-necked flask, under magnetic agitation after mixing, is slowly added to 1.0 mol/L NaOH and adjusts pH to 11, in N2Under protection
After stirring the oxygen that 30min is removed in mixed liquor, 0.1 mol/L Na of 5mL is added dropwise2S•9H2O aqueous solution continues in N2It protects
Shield is lower to stir 4h, and water-soluble CdS metal quantum point solution can be obtained；
3, the production of nicotine working curve: by 1,20,40,60,80, the nicotine standard solution 0.5mL of 100mg/L is transferred to
In 10mL color-comparison tube, 20 μ L water-soluble metal quantum dot solutions, 60 μ L Water-soluble carbon quantum dot solution are sequentially added, pH is used
6.0 phosphate buffer solutions (being made of sodium dihydrogen phosphate and disodium hydrogen phosphate) are diluted to scale, shake up, and stand 8min, with
300nm is excitation wavelength, and excitation and transmite slit are 5nm, and the fluorescence intensity F measured at 600nm obtains regression equation
(F-F0)/F0 = 0.2187C (μg mL-1)+0.0809, correlation coefficient r=0.9991, F indicates that metal quantum point is deposited in nicotine
When fluorescence intensity, F0Respectively indicate fluorescence intensity of metal quantum point in the presence of no nicotine；
4, sample treatment: the cigarette cut tobacco 1.000g for accurately weighing certain commercially available brand is placed in 100 mL flasks, is added
100 mL dehydrated alcohols impregnate for 24 hours, separate filtrate, add 100 mL soaked in absolute ethyl alcohol, 24 h, filter, merging filtrate,
Constant volume obtains nicotine extracting solution in 250mL volumetric flask；
5, in cigarette sample nicotine measurement: removing step 4 prepare nicotine extracting solution 5mL, be transferred to 10mL tool plug ratio
In colour tube, 20 μ L water-soluble metal quantum dot solutions, 60 μ L Water-soluble carbon quantum dot solution are added, with pH6.0 phosphate-buffered
Solution (being made of sodium dihydrogen phosphate and disodium hydrogen phosphate) is diluted to scale, shakes up, and stands 8min, using 300nm as excitation wavelength,
Excitation and transmite slit are 5nm, and the fluorescence intensity F measured at 600nm substitutes into step 3 regression equation, calculate sample nicotine
Content is 13.1 mg/g.
Embodiment 2: the measurement of nicotine in tobacco juice for electronic smoke
1, the preparation of Water-soluble carbon quantum dot solution: weighing 10.0g polyethyleneimine liquid and be added in 90mL ultrapure water,
Ultrasonic 8min mixes well it, and is transferred to ptfe autoclave, after 180 DEG C of heating 7h, cooled to room temperature,
Be first 0.22 μm of membrane filtration with aperture, after with the bag filter that molecular cut off is 3000Da carry out dialysis treatment for 24 hours, obtain
Water-soluble carbon quantum dot solution；
2, the preparation of water-soluble metal quantum dot: by 0.01 mol/L CdCl of 60mL2Solution, 300 μ L thioacetic acid add
Enter in three-necked flask, under magnetic agitation after mixing, is slowly added to 1.0 mol/L NaOH and adjusts pH to 11, in N2Under protection
After stirring the oxygen that 40min is removed in mixed liquor, 5mL 0.2mol/L Na is added dropwise2S•9H2O aqueous solution continues in N2It protects
Shield is lower to stir 4h, and water-soluble CdS metal quantum point solution can be obtained；
3, the production of nicotine working curve: by 1,20,40,60,80, the nicotine standard solution 0.5mL of 100mg/L is transferred to
In 10mL color-comparison tube, 30 μ L water-soluble metal quantum dot solutions, 70 μ L Water-soluble carbon quantum dot solution are sequentially added, pH is used
6.2 phosphate buffer solutions are diluted to scale, shake up, and stand 9min, and using 300nm as excitation wavelength, excitation and transmite slit are equal
Regression equation (F-F is obtained for 5nm, the fluorescence intensity F measured at 600nm0)/F0 = 0.0298C (μg mL-1) +
0.0301, correlation coefficient r=0.9990, F and F0, fluorescence intensity of F expression metal quantum point in the presence of nicotine, F0It respectively indicates
Fluorescence intensity of metal quantum point in the presence of no nicotine；
4, sample treatment: weighing tobacco juice for electronic smoke 0.1g sample, is accurate to 0.0001g, and it is dense that 10mL percent by volume is added
Degree 10% isopropanol water solution, vortex mixed 1min, be centrifugated (being centrifuged 10min under 3000r/ min), and take centrifugation after
Isopropanol water solution layer clear liquid is as nicotine extracting solution；
5, in cigarette sample nicotine measurement: removing step 4 prepare nicotine extracting solution 5mL, be transferred to 10mL tool plug ratio
In colour tube, 30 μ L water-soluble metal quantum dot solutions, 70 μ L Water-soluble carbon quantum dot solution are added, with 6.2 phosphate-buffered of pH
Solution is diluted to scale, shakes up, and stands 9min, and using 300nm as excitation wavelength, excitation and transmite slit are 5nm, in 600nm
Locate the fluorescence intensity F of measurement, substitute into step 3 regression equation, calculating sample nicotine content is 15.6 mg/g.
Embodiment 3: the measurement of nicotine in tobacco sample
1, the preparation of Water-soluble carbon quantum dot solution: weighing 10.0g polyethyleneimine liquid and be added in 90mL ultrapure water,
Ultrasonic 6min mixes well it, and is transferred to ptfe autoclave, after 190 DEG C of heating 6h, cooled to room temperature,
Be first 0.22 μm of membrane filtration with aperture, after with the bag filter that molecular cut off is 3200Da carry out dialysis treatment for 24 hours, obtain
Water-soluble carbon quantum dot solution；
2, the preparation of water-soluble metal quantum dot: by 0.01 mol/L CdCl of 70mL2Solution, 260 μ L thioacetic acid add
Enter in three-necked flask, under magnetic agitation after mixing, is slowly added to 1.0 mol/L NaOH and adjusts pH to 11, in N2Under protection
After stirring the oxygen that 45min is removed in mixed liquor, 5mL 0.15mol/L Na is added dropwise2S•9H2O aqueous solution continues in N2It protects
Shield is lower to stir 4h, and water-soluble CdS metal quantum point solution can be obtained；
3, the production of nicotine working curve: by 1,20,40,60,80, the nicotine standard solution 0.5mL of 100mg/L is transferred to
In 10mL color-comparison tube, 40 μ L water-soluble metal quantum dot solutions, 80 μ L Water-soluble carbon quantum dot solution are sequentially added, pH is used
6.5 phosphate buffer solutions are diluted to scale, shake up, and stand 10min, and using 300nm as excitation wavelength, excitation and transmite slit are equal
Regression equation (F-F is obtained for 5nm, the fluorescence intensity F measured at 600nm0)/F0 = 0.1203C (μg mL-1) +
0.1230, correlation coefficient r=0.9992, F indicates fluorescence intensity of metal quantum point in the presence of nicotine, F0Respectively indicate metal
Fluorescence intensity of quantum dot in the presence of no nicotine；
4, sample treatment: accurately weighing dry tobacco leaf powder 1.000g and be placed in a beaker, and it is molten that 0.5mol/L HCl is added
Liquid 30mL, slightly boiled 10min, filters while hot in small fire, reusable heat water washing residue 3 times, and filtrate merges, and 0.5mol/L is added
NaOH solution tune pH to 7 is settled in 250mL volumetric flask with water and obtains nicotine extracting solution；
5, in cigarette sample nicotine measurement: removing step 4 prepare nicotine extracting solution 5mL, be transferred to 10mL tool plug ratio
In colour tube, 40 μ L water-soluble metal quantum dot solutions, 80 μ L Water-soluble carbon quantum dot solution are added, with 6.5 phosphate-buffered of pH
Solution is diluted to scale, shakes up, and stands 10min, and using 300nm as excitation wavelength, excitation and transmite slit are 5nm, in 600nm
Locate the fluorescence intensity F of measurement, substitute into step 3 regression equation, calculating sample nicotine content is 9.2 mg/g.
Embodiment 1- embodiment 3 is subjected to recovery of standard addition and relative standard deviation test, and by the method for the present invention in
The measuring method of state tobacco professional standard YC/T 468-2013 tobacco and tobacco product nicotine is compared, and the results are shown in Table 1；
1 recovery of standard addition of table and relative standard deviation (n=5)
As seen from the results in Table 1: with the method and YC/T 246-2008 of fluorescence resonance energy transfer measurement nicotine of the invention
Measuring method is compared, and as a result unanimously, but because processing step is few, the time used is short, and processing cost is low, and consumption of organic solvent has more less
Advantage can be seen that the rate of recovery between 93.8-102.1% from high, normal, basic recovery of standard addition, between relative standard deviation 1.0-2.8%,
The accuracy and precision that illustration method has had.
Fig. 1-3 confirms the condition that fluorescence resonance energy transfer provided by the invention occurs and turns to fluorescence resonance energy is established
Shifting system；From figure 1 it appears that a length of 256 nm of the maximum absorption wave of carbon quantum dot, maximum emission wavelength is 440 nm, gold
Belong to a length of 423 nm of maximum absorption wave of quantum dot, maximum emission wavelength is 600 nm；Meet fluorescence resonance energy transfer
Condition, using carbon quantum dot as donor, metal quantum point is receptor, the launch wavelength of carbon quantum dot and the absorption wave of metal quantum point
Long overlapping degree is high, and transferring efficiency of fluorescence resonance energy is high, has the condition of resonance energy transfer；Fig. 2 shows when fixed gold
Belong to quantum dot concentration, changes carbon quantum dot concentration, metal quantum point fluorescence intensity increases, and corresponding to carbon quantum dot fluorescence has quenching
Phenomenon occurs, this is because energy has been transferred to receptor by donor, it was demonstrated that the presence of fluorescence resonance system；Fig. 3 shows to work as fixation
The fluorescence intensity of the concentration of carbon quantum dot, metal quantum point enhances with the increase of carbon quantum dot concentration, is due to more
Energy donor is transferred to receptor, further demonstrates the foundation of fluorescence resonance energy system.
Fig. 4 is the foundation of nicotine quantitative analysis, and Fig. 4 shows that nicotinic density variation is linear with metal quantum point fluorescence intensity
Relationship has measurement nicotine condition, provides the support of nicotine analysis.
Claims (3)
1. a method of nicotine is detected based on fluorescence resonance energy transfer, comprising the following steps:
(1) 20~40 μ L water-soluble metal quantum dots the production of nicotine working curve: are sequentially added in 10mL color-comparison tube
Solution, 60~80 μ L Water-soluble carbon quantum dot solution and 0.5 mL nicotine standard solution are diluted with 6.0~6.5 buffer solution of pH
It to scale, shakes up, stands 8~10min, using 300nm as excitation wavelength, excitation and transmite slit are 5nm, measure 600nm's
Fluorescence intensity F, calculates (F-F0)/F0Regression equation, F indicates fluorescence intensity of metal quantum point in the presence of nicotine, F0Table
Show fluorescence intensity of metal quantum point in the presence of no nicotine；
(2) sample treatment
Cigarette sample: accurately weighing cigarette cut tobacco 1.000g and be placed in flask, and 80~120 mL dehydrated alcohols are added, and impregnates 12
~for 24 hours, filtrate is separated, adds 80~120 mL soaked in absolute ethyl alcohol 12~for 24 hours, filtering, merging filtrate, constant volume is in 250 mL
In volumetric flask, nicotine extracting solution is obtained；
Tobacco juice for electronic smoke: in the ratio of 1.000g tobacco juice for electronic smoke addition 100~150mL isopropanol water solution, by electronic cigarette
Tobacco juice is dissolved in isopropanol water solution, and the concentration of volume percent of isopropanol water solution is 5~15%, 1~3min of vortex mixed,
Centrifuge separation, and take the clear liquid after centrifugation as nicotine extracting solution；
Tobacco sample: accurately weighing dry tobacco leaf powder 1.000g and be placed in a beaker, and 0.5~1.0 mol/L HCl is added
30~50 mL of solution, slightly boiled 10~15min, filters while hot in small fire, reusable heat water washing residue 3~4 times, and filtrate is closed
And 0.5~1.0 mol/L NaOH solution tune pH to 7 is added, it is settled in 250mL volumetric flask with water and obtains nicotine extracting solution；
(3) sample measures: 20~40 μ L water-soluble metal quantum dot solutions, 60~80 are sequentially added in 10mL color-comparison tube
μ L Water-soluble carbon quantum dot solution and the nicotine extracting solution of 5mL step (2) preparation, are diluted to 6.0~6.5 buffer solution of pH
Scale shakes up, and stands 8~10min, and using 300nm as excitation wavelength, excitation and transmite slit are 5nm, measures the glimmering of 600nm
Luminous intensity F, calculates (F-F0)/F0, step (1) regression equation is substituted into, sample nicotine content is calculated；
The Water-soluble carbon quantum dot solution the preparation method comprises the following steps: weighing 10.0g polyethyleneimine liquid, to be added to 90mL ultrapure
In water, 5~8min of ultrasound mixes well it, and is transferred to ptfe autoclave, in 180~200 DEG C of 5~7h of heating,
It is first 0.22 μm of membrane filtration with aperture after cooled to room temperature, is then the saturating of 3000~3500Da with molecular cut off
It analyses bag and carries out dialysis treatment for 24 hours, obtain Water-soluble carbon quantum dot solution；
The water-soluble metal quantum dot solution the preparation method comprises the following steps: by 50~70 mL, 0.01 mol/L CdCl2Solution, 250
~300 μ L thioacetic acid are added in three-necked flask, under magnetic agitation after mixing, are slowly added to 1.0 mol/L NaOH adjusting
PH to 11, in N2After 30~45min of the lower stirring of protection removes the oxygen in mixed liquor, 0.1~0.2 mol/L is added dropwise
Na2S•9H2O aqueous solution 5mL continues in N2Protection is lower to stir 4h, and water-soluble CdS metal quantum point solution can be obtained.
2. the method according to claim 1 based on fluorescence resonance energy transfer detection nicotine, it is characterised in that: pH 6.0
The buffer solution that~6.5 buffer solutions are made of sodium dihydrogen phosphate and disodium hydrogen phosphate.
3. the method according to claim 1 based on fluorescence resonance energy transfer detection nicotine, it is characterised in that: centrifugation point
From be at 3000~6000r/ of centrifugation rate min be centrifuged 5~10min.
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| CN112683864A (en) * | 2020-12-11 | 2021-04-20 | 广西中烟工业有限责任公司 | Rapid detection method for nicotine in flue-cured tobacco leaves |
| CN113720822A (en) * | 2021-09-18 | 2021-11-30 | 广西中烟工业有限责任公司 | Method for detecting nicotine in electronic cigarette by combining magnetic solid phase extraction with carbon dot fluorescent probe |
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