CN103472046A - Method of analyzing heavy metal pollution of vegetable based on reactive oxygen species level - Google Patents
Method of analyzing heavy metal pollution of vegetable based on reactive oxygen species level Download PDFInfo
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- CN103472046A CN103472046A CN2013104404781A CN201310440478A CN103472046A CN 103472046 A CN103472046 A CN 103472046A CN 2013104404781 A CN2013104404781 A CN 2013104404781A CN 201310440478 A CN201310440478 A CN 201310440478A CN 103472046 A CN103472046 A CN 103472046A
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Abstract
The invention provides a method of evaluating the heavy metal pollution of vegetable based on reactive oxygen species level by analyzing the fluorescence intensity of reactive oxygen species in vegetable cells through adopting laser scanning confocal microscope technology. The method comprises the following steps: 1) marking reactive oxygen species in vegetable leaf cells by adopting 2',7'-dichorofluorescein diacetate (DCFH-DA); 2) analyzing the reactive oxygen species level in vegetable leaf cells by adopting the laser scanning confocal microscope technology; 3) fixing instrument acquisition parameters by taking the fluorescence intensity of reactive oxygen species in the non-polluted vegetable leaves according with the national standard; 4) further acquiring the fluorescence density value of the vegetable leaf to be detected according to the instrument parameter, performing statistic analysis on the fluorescence intensity value of vegetable to be detected and the standard vegetable fluorescence intensity value so as to confirm whether the vegetable is polluted by heavy metal.
Description
Technical field
The present invention relates to a kind of method based on active oxygen radical horizontal analysis Contamination of Heavy Metal in Vegetables, belong to the food inspection technical field.
Background technology
Along with the fast development of industrial or agricultural, the discharge of Industrial " three Waste " and a large amount of uses of the material used for agriculture such as agricultural chemicals, chemical fertilizer, make vegetables be subject to heavy metal pollution in various degree.Soil microorganism can not be decomposed heavy metal, heavy metal is enrichment in vivo, some heavy metal will be transformed into the methyl compound that toxicity is larger in vivo, and these pollutants are likely accumulated to be harmful to concentration by food chain in human body, the serious harm health.Therefore, be necessary Heavy Metals In Vegetables is analyzed and estimated, for guaranteeing vegetables quality and ensureing that people's safe diet is significant.
Liquid chromatography, gas chromatography and spectral technique are to analyze the whether contaminated conventional method of analysis of vegetables, but these method sample pre-treatments are comparatively complicated, and routine analyzer is loaded down with trivial details.Laser scanning co-focusing microscope is to install laser scanning device additional on the basis of fluorescent microscope imaging, uses ultraviolet light or excited by visible light fluorescence probe, utilizes computing machine to carry out the image processing.Utilize laser scanning co-focusing microscope can process sample alive, can not cause the destruction of physicochemical characteristics to sample, more approach the cell life state parameter and measure.But laser scanning co-focusing microscope technology express-analysis plant sample, without loaded down with trivial details sample pretreatment process.
Fluorescence microscopic analysis technology and fluorescence analyzer are widely used in plant cytology research, and single molecule fluorescence detection has considerable advantage in the research of living cells system.Can measure Cytoplasmic Ca by the fluoroscopic image analysis
2+, cell membrane potential, mitochondrial membrane potential, cell membrane phospholipid and acceptor mobility.Active oxygen radical refers to by the general name of several oxygen carriers that oxygen forms, chemical property is active, oxidability is very strong, comprises singlet oxygen, hydroxy radical, superoxide anion, hydrogen peroxide etc.Under normal circumstances, activity in vivo oxygen produces and removes in equilibrium state plant.Once and be subject to heavy metal stress, this balance will be destroyed, the active oxygen radical level rises.In vegetables, active oxygen radical is in the certain level scope, once and vegetables are polluted by heavy metal or other adjuvants, the active oxygen radical level will change.
2 ', 7 '-dichlorofluorescein diacetate (DCFH-DA) itself does not have fluorescence, can pass freely through cell membrane, after entering cell, can be generated DCFH by intracellular esterase hydrolyzed.Intracellular active oxygen can generate the DCF that fluorescence is arranged by the non-blooming DCFH of oxidation, by detecting DCF fluorescence to judge the level of reactive oxygen species.In vegetables, the active oxygen radical fluorescence intensity is in the certain level scope, once and vegetables are subject to heavy metal pollution, the active oxygen radical fluorescence intensity will change.The present invention adopts DCFH-DA mark vegetables intracellular reactive oxygen radical, adopts laser scanning co-focusing microscope technical Analysis vegetables intracellular reactive oxygen free radicals.In actual testing process, only need to gather to meet active oxygen radical fluorescence intensity in the not comtaminated vegetable leaf of national standard (standard fluorescence intensity level), the retainer instrument acquisition parameter, vegetables fluorescence intensity level and vegetables standard fluorescence intensity level that needs are detected are compared, and statistical analysis just can determine whether vegetables are subject to heavy metal pollution.The present invention adopts laser scanning co-focusing microscope technical Analysis vegetables intracellular reactive oxygen radical fluorescence intensity, has set up the Contamination of Heavy Metal in Vegetables evaluation method based on the active oxygen radical level.
Summary of the invention
The purpose of this invention is to provide a kind of method based on active oxygen radical assessment of levels Contamination of Heavy Metal in Vegetables.
The purpose of this invention is to provide a kind of method based on active oxygen radical assessment of levels Contamination of Heavy Metal in Vegetables, comprise the steps: 1) adopt 2 ', 7 '-dichlorofluorescein diacetate (DCFH-DA) mark vegetable leaf intracellular reactive oxygen radical; 2) adopt laser scanning co-focusing microscope technical Analysis vegetable leaf intracellular reactive oxygen free radicals; 3) will meet in the not comtaminated vegetable leaf of national standard the active oxygen radical fluorescence intensity as the standard fluorescence intensity level, the retainer instrument acquisition parameter; 4) further gather and need to detect fluorescence intensity level in vegetable leaf according to above-mentioned instrument parameter, vegetables fluorescence intensity level and vegetables standard fluorescence intensity level that needs are detected carry out statistical study, determine whether vegetables are subject to heavy metal pollution.
technical scheme
Experimental principle: 2 ', 7 '-dichlorofluorescein diacetate (DCFH-DA) itself does not have fluorescence, can pass freely through cell membrane, after entering cell, can be generated DCFH by intracellular esterase hydrolyzed.Intracellular active oxygen can generate the DCF that fluorescence is arranged by the non-blooming DCFH of oxidation, by detecting DCF fluorescence to judge the level of reactive oxygen species.In vegetables, the active oxygen radical fluorescence intensity is in the certain level scope, once and vegetables are subject to heavy metal pollution, the active oxygen radical fluorescence intensity will change.
Vegetable leaf disposal route: get fresh not comtaminated certain vegetable leaf and (measure through icp ms, this vegetables content of beary metal meets national standard), rinse well with isotonic buffer solution (0.5 mol/L D-sorbite, 4 mol/L MESs, 20 mmol/L potassium chloride, pH 5.7), carefully cut slender blade with blade some.Subsequently, blade is proceeded to 10 μ mol/L 2 ' 7 '-dichlorofluorescein diacetates (DCFH-DA) and carry out active oxygen dyeing 2 hours, isotonic buffer solution rinses.Vegetable leaf after rinsing with the tweezers gripping subsequently, put into microslide.
Active oxygen radical image acquisition and instrument parameter are set up: adopt laser confocal microscope to obtain fluoroscopic image, the Argon ion laser excitation wavelength is 488 nanometers, and it is 500 ~ 650 nanometers that signal receives wavelength, and it is green gathering fluoroscopic image.While gathering different vegetable active oxygen in leaves free radical fluorescence picture, instrument parameter stores, and all blades all adopt identical parameters.Subsequently, adopt Leica TCS-SP 2 softwares to carry out active oxygen radical fluorescence statistical study in vegetable leaf.The heavy metal of each concentration is processed 10 blades of statistics, analyzes blade cell active oxygen radical fluorescence intensity, and this fluorescence intensity level stores as the standard fluorescence intensity level.
Vegetable Sold is analyzed: get commercially available this vegetable leaf, disposal route is same as above, adopt identical instrument parameter to gather vegetable leaf active oxygen radical fluorescence picture, subsequently, adopt Leica TCS-SP 2 softwares to carry out active oxygen radical fluorescence statistical study in vegetable leaf.The heavy metal of each concentration is processed 10 blades of statistics, analyzes blade cell active oxygen radical fluorescence intensity.
The Contamination of Heavy Metal in Vegetables determination methods: adopt statistical software to carry out statistical study to data, statistical treatment adopts the t method of inspection, and vegetable active oxygen radical fluorescence intensity level and standard fluorescence intensity level that needs are detected carry out significance test.P<0.05 means to need to detect vegetable active oxygen radical fluorescence intensity level and there is significant difference in the standard fluorescence intensity level, and P<0.01 means to exist utmost point significant difference, P<0.05 or P<0.01 all to mean that these vegetables are subject to heavy metal pollution.
Embodiment
Embodiment 1
1 icp ms measures content of beary metal and the standard vegetables are set: the lead ion standard solution is purchased from chemi-ionization institute of Shanghai Institute of Measurement and Testing Technology, getting concentration known is the plumbous standard solution of 1000 μ g/mL, add appropriate nitric acid, be mixed with the standard solution of gradient with the ultrapure water dilution, obtain plumbous standard solution series after shaking up.
Adopt icp ms (U.S. Agilent HP4500 Series 300) to measure content of beary metal, by the optimization to the icp ms running parameter, set up the quantitative analysis method of lead element content in vegetable leaf.
Adopt icp ms to measure commercially available spinach content of heavy metal lead, in three portions of spinach, lead ion content is respectively 40.26 ± 1.26,82.35 ± 2.02,160.58 ± 1.04 μ g/Kg, content be 40.26 ± 1.26 μ g/Kg lower than national standard, using it as the standard vegetables further to measure its standard fluorescence intensity level.
2 active oxygen radical picture collections and fluorescence intensity analysis: get above-mentioned three parts of spinach vegetable leafs, rinse well with isotonic buffer solution (0.5 mol/L D-sorbite, 4 mol/L MESs, 20 mmol/L potassium chloride, pH 5.7), carefully cut slender blade with blade some.Subsequently, blade is proceeded to 10 μ mol/L 2 ' 7 '-dichlorofluorescein diacetates (DCFH-DA) and carry out active oxygen dyeing 2 hours, isotonic buffer solution rinses.Vegetable leaf after rinsing with the tweezers gripping subsequently, put into microslide.
Adopt laser scanning co-focusing microscope (Leca company produces, model Leica TCS-SP 2) to obtain fluoroscopic image, the Argon ion laser excitation wavelength is 488 nanometers, and it is 500 ~ 650 nanometers that signal receives wavelength, and it is green gathering fluoroscopic image.While gathering different vegetable active oxygen in leaves free radical fluorescence picture, instrument parameter stores, and all blades all adopt identical parameters.Subsequently, adopt Leica TCS-SP 2 softwares to carry out active oxygen radical fluorescence statistical study in vegetable leaf.The heavy metal of each concentration is processed 10 blades of statistics, analyzes blade cell active oxygen radical fluorescence intensity.Active oxygen radical standard fluorescence intensity level in the bioassay standard vegetable leaf, and further analyze other two parts of vegetable active oxygen radical fluorescence intensity levels.
3 interpretations of result: adopt laser scanning co-focusing microscope to carry out active oxygen radical analysis mensuration to three parts of Spinach Leafs.Standard vegetables lead ion content is 40.26 ± 1.26 μ g/Kg, and standard vegetable leaf active oxygen radical fluorescence intensity level is 86.34 ± 12.72; Other two parts of vegetables lead ion contents are respectively 82.35 ± 2.02 μ g/Kg, 160.58 ± 1.04 μ g/Kg, the active oxygen radical fluorescence intensity level is respectively 98.96 ± 13.24,110.36 ± 11.23, and the active oxygen radical fluorescence intensity corresponding with three kinds of plumbum ion concentrations is in rising trend.Result shows, content of heavy metal lead raises, and the two kinds of vegetable active oxygen radical fluorescence intensity levels to be measured all utmost point are significantly higher than standard fluorescence intensity level (P<0.01), can judge that accordingly vegetables are polluted by heavy metal lead.
Embodiment 2
1 icp ms measures content of beary metal and the standard vegetables are set: the mercury ion standard solution is purchased from chemi-ionization institute of Shanghai Institute of Measurement and Testing Technology, get the mercury standard solution that concentration known is 1000 μ g/mL, add appropriate nitric acid, be mixed with the standard solution of gradient with the ultrapure water dilution, obtain mercury standard solution series after shaking up.
Adopt icp ms (U.S. Agilent HP4500 Series 300) to measure content of beary metal, by the optimization to the icp ms running parameter, set up the quantitative analysis method of mercury element content in vegetable leaf.
Adopt icp ms to measure commercially available spinach heavy metal Hg content, in three portions of spinach, mercury ion content is respectively 2.26 ± 0.31,10.35 ± 0.35,20.58 ± 0.62 μ g/Kg, content be 2.26 ± 0.31 μ g/Kg lower than national standard, using it as the standard vegetables further to measure its standard fluorescence intensity level.
2 active oxygen radical picture collections and fluorescence intensity analysis: get above-mentioned three parts of spinach vegetable leafs, rinse well with isotonic buffer solution (0.5 mol/L D-sorbite, 4 mol/L MESs, 20 mmol/L potassium chloride, pH 5.7), carefully cut slender blade with blade some.Subsequently, blade is proceeded to 10 μ mol/L 2 ' 7 '-dichlorofluorescein diacetates (DCFH-DA) and carry out active oxygen dyeing 2 hours, isotonic buffer solution rinses.Vegetable leaf after rinsing with the tweezers gripping subsequently, put into microslide.
Adopt laser scanning co-focusing microscope (Leca company produces, model Leica TCS-SP 2) to obtain fluoroscopic image, the Argon ion laser excitation wavelength is 488 nanometers, and it is 500 ~ 650 nanometers that signal receives wavelength, and it is green gathering fluoroscopic image.While gathering different vegetable active oxygen in leaves free radical fluorescence picture, instrument parameter stores, and all blades all adopt identical parameters.Subsequently, adopt Leica TCS-SP 2 softwares to carry out active oxygen radical fluorescence statistical study in vegetable leaf.The heavy metal of each concentration is processed 10 blades of statistics, analyzes blade cell active oxygen radical fluorescence intensity.Active oxygen radical standard fluorescence intensity level in the bioassay standard vegetable leaf, and further analyze other two parts of vegetable active oxygen radical fluorescence intensity levels.
3 interpretations of result: adopt laser scanning co-focusing microscope to carry out active oxygen radical analysis mensuration to three parts of Spinach Leafs.Standard vegetables mercury ion content is 2.26 ± 0.31 μ g/Kg, and standard vegetable leaf active oxygen radical fluorescence intensity level is 80.82 ± 2.19; Other two parts of vegetables mercury ion content are respectively 10.35 ± 0.35 μ g/Kg, 20.58 ± 0.62 μ g/Kg, the active oxygen radical fluorescence intensity level is respectively 89.76 ± 2.49,97.38 ± 1.32, and the active oxygen radical fluorescence intensity corresponding with three kinds of ion concentration of mercury is in rising trend.Result shows, heavy metal Hg content raises, and the two kinds of vegetable active oxygen radical fluorescence intensity levels to be measured all utmost point are significantly higher than standard fluorescence intensity level (P<0.01), can judge that accordingly vegetables are subject to heavy metal pollution.
Embodiment 3
1 icp ms measures content of beary metal and the standard vegetables are set: the cadmium ion standard solution is purchased from chemi-ionization institute of Shanghai Institute of Measurement and Testing Technology, getting concentration known is the cadmium standard solution of 1000 μ g/mL, add appropriate nitric acid, be mixed with the standard solution of gradient with the ultrapure water dilution, obtain cadmium standard solution series after shaking up.
Adopt icp ms (U.S. Agilent HP4500 Series 300) to measure content of beary metal, by the optimization to the icp ms running parameter, set up the quantitative analysis method of cadmium element content in vegetable leaf.
Adopt icp ms to measure commercially available spinach heavy metal cadmium content, in three portions of spinach, cadmium ion content is respectively 5.76 ± 0.25,15.32 ± 0.65,25.67 ± 0.43 μ g/Kg, content be 5.76 ± 0.25 μ g/Kg lower than national standard, using it as the standard vegetables further to measure its standard fluorescence intensity level.
2 active oxygen radical picture collections and fluorescence intensity analysis: get above-mentioned three parts of spinach vegetable leafs, rinse well with isotonic buffer solution (0.5 mol/L D-sorbite, 4 mol/L MESs, 20 mmol/L potassium chloride, pH 5.7), carefully cut slender blade with blade some.Subsequently, blade is proceeded to 10 μ mol/L 2 ' 7 '-dichlorofluorescein diacetates (DCFH-DA) and carry out active oxygen dyeing 2 hours, isotonic buffer solution rinses.Vegetable leaf after rinsing with the tweezers gripping subsequently, put into microslide.
Adopt laser scanning co-focusing microscope (Leca company produces, model Leica TCS-SP 2) to obtain fluoroscopic image, the Argon ion laser excitation wavelength is 488 nanometers, and it is 500 ~ 650 nanometers that signal receives wavelength, and it is green gathering fluoroscopic image.While gathering different vegetable active oxygen in leaves free radical fluorescence picture, instrument parameter stores, and all blades all adopt identical parameters.Subsequently, adopt Leica TCS-SP 2 softwares to carry out active oxygen radical fluorescence statistical study in vegetable leaf.The heavy metal of each concentration is processed 10 blades of statistics, analyzes blade cell active oxygen radical fluorescence intensity.Active oxygen radical standard fluorescence intensity level in the bioassay standard vegetable leaf, and further analyze other two parts of vegetable active oxygen radical fluorescence intensity levels.
3 interpretations of result: adopt laser scanning co-focusing microscope to carry out active oxygen radical analysis mensuration to three parts of Spinach Leafs.Standard vegetables cadmium ion content is 5.76 ± 0.25 μ g/Kg, and standard vegetable leaf active oxygen radical fluorescence intensity level is 83.56 ± 1.62; Other two parts of vegetables cadmium ion content are respectively 15.32 ± 0.65 μ g/Kg, 25.67 ± 0.43 μ g/Kg, the active oxygen radical fluorescence intensity level is respectively 91.78 ± 2.14,103.22 ± 5.21, and the active oxygen radical fluorescence intensity corresponding with three kinds of concentration of cadmium ions is in rising trend.Result shows, heavy metal cadmium content raises, and the two kinds of vegetable active oxygen radical fluorescence intensity levels to be measured all utmost point are significantly higher than standard fluorescence intensity level (P<0.01), can judge that accordingly vegetables are subject to heavy metal pollution.
Claims (4)
1. the method based on active oxygen radical horizontal analysis Contamination of Heavy Metal in Vegetables, is characterized in that adopting laser scanning co-focusing microscope technical Analysis vegetables whether to be subject to heavy metal pollution and vegetable safety.
2. the method based on active oxygen radical horizontal analysis Contamination of Heavy Metal in Vegetables, is characterized in that whether being subject to heavy metal pollution and vegetable safety according to active oxygen radical assessment of levels vegetables.
3. the method based on active oxygen radical horizontal analysis Contamination of Heavy Metal in Vegetables, is characterized in that collection meets active oxygen radical fluorescence intensity in the not comtaminated vegetable leaf of national standard, using it as the standard fluorescence intensity level.
4. the method based on active oxygen radical horizontal analysis Contamination of Heavy Metal in Vegetables, is characterized in that vegetables fluorescence intensity level and the vegetables standard fluorescence intensity level of needs detection are compared, and according to statistic analysis result, determines whether vegetables are subject to heavy metal pollution.
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| CN107084957A (en) * | 2016-06-29 | 2017-08-22 | 南京大学 | A kind of method detected to intracellular reactive oxygen content |
| CN114200065A (en) * | 2021-12-08 | 2022-03-18 | 维塔探索(广东)科技有限公司 | Method for quantitatively measuring content of in vivo/in vitro superoxide radical generated by environmental stress |
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2013
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107084957A (en) * | 2016-06-29 | 2017-08-22 | 南京大学 | A kind of method detected to intracellular reactive oxygen content |
| CN107084957B (en) * | 2016-06-29 | 2020-09-08 | 南京大学 | Method for detecting content of active oxygen in cells |
| CN106520904A (en) * | 2016-11-25 | 2017-03-22 | 云南中烟工业有限责任公司 | Method for detecting influence of total particulate matters in cigarette smoke on reactive oxygen secretion of cells |
| CN106908421A (en) * | 2017-01-18 | 2017-06-30 | 北京蓝色星语科技有限公司 | A kind of multichannel dangerous substance detection method and detection means |
| CN114200065A (en) * | 2021-12-08 | 2022-03-18 | 维塔探索(广东)科技有限公司 | Method for quantitatively measuring content of in vivo/in vitro superoxide radical generated by environmental stress |
| CN114200065B (en) * | 2021-12-08 | 2022-07-26 | 维塔探索(广东)科技有限公司 | Method for quantitatively measuring content of in vivo/in vitro superoxide radical generated by environmental stress |
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