CN102297875B - Method for measuring hydroxyl free radical in plant body - Google Patents
Method for measuring hydroxyl free radical in plant body Download PDFInfo
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Abstract
The invention relates to a method for utilizing electron spin resonance (ESR) and secondary free radical spin acquiring processes to measure a hydroxyl free radical in a plant body. The method comprises the following steps: taking dimethyl sulfoxide (DMSO) as a first free radical capturing agent; taking N-tertiary butyl-alpha-Nitrate ketone benzene (PBN) as a secondary free radical capturing agent; and after performing a series of capturing and extracting processes, utilizing ESR to measure the hydroxyl free radical in a plant sample.
Description
Technical field
The present invention relates to the assay method of free radical, specifically use electron spin resonance in conjunction with secondary free radical spin trapping technology, by catching, extracting, with electron spin paramagnetic resonance, measure the method for the hydroxy free radical concentration in plant sample.
Background technology
Soil pollution is one of worldwide environmental problem.Approximately 2,000 ten thousand hectares of China's heavy-metal contaminated soils, ten thousand hectares, agricultural chemicals, microbiotic, pathogen contamination soil 1300-1600, approximately 2,000 ten thousand hectares, the more serious arable land of damage ratio, accounts for 1/5 of cultivated area.Soil pollution has a negative impact to crop yield, quality.China because of more than 1,000 ten thousand tons, soil pollution underproduction grain, causes approximately 20,000,000,000 yuan of economic losses every year.Therefore, clean and the safe utilization of contaminated soil is an important task, and soil pollution diagnosis is one of them important step.The simple chemical method that relies on is carried out soil pollution diagnosis, can not characterize the total quality feature of soil comprehensive, scientifically, need additive method to make this supplementary, the ecological toxicity diagnosis research of soil pollution is complied with this objective requirement, has obtained international extensive attention and has developed rapidly.
Higher plant is the element in soil ecosystem.Utilize its upgrowth situation, thereby from organizing level, molecular level judgement contaminated soil can reach the object of diagnosis soil pollution to the impact of plant growth.Biology there will be various free radicals in its aerobic metabolism process, in biological cell, there are again various radicals scavenging systems simultaneously, thereby in cell, free radical maintains rational level, but under as adverse environmental factors such as environmental pollution, herbicide uses, in biosome, often there is the free radical of excess and biological metabolism is exerted an influence.Research data shows, the free radical producing in biosome mainly comprises superoxide radical, hydroxy radical (OH), lipid peroxidation product, hydrogen peroxide (H
2o
2), singlet oxygen (
1o
2) etc. oxygen radical and nitrogen monoxide (NO
2) etc. nitrogen free radical etc.This part free radical forming in biosome will cause lipid peroxidation, protein denaturation, nucleic acid molecules and gene mutation, the fracture of carbohydrates carbochain of cell inner membrance etc.Therefore according to the variation of Kinds of Free Radicals in plant and intensity, can judge that whether plant is in oxidative stress status, for pollutant provides the most direct evidence to the murder by poisoning Mechanism Study of plant and the early diagnosis of contaminated soil.
It is the active oxygen species of tool activity in biosystem that hydroxyl radical free radical (OH) is recognized, and it has extremely strong oxidability, and can cause induction and produce chain reaction.Thus, development accurately, sensitive OH detection technique is very important.Traditional OH chemical detection, as highly sensitive, easy to operate in spectrophotometric method, fluorimetry, chemoluminescence method etc., but disturbing factor in mensuration process is more.Electron spin resonance is the most frequently used, the most effective detection method, and it has the advantages such as quick, easy, economy, consumption are few, has now been widely used in biology, medical research field.The life-span of OH is extremely short, only has 10
-6second, so electron spin resonance need coordinate trapping agent, to extend the life-span of free radical.Its principle is to utilize suitable spin trapping agent and active short life combined with radical, generate metastable spin adduct, then with electron spin resonance, detect the quantity of spin adduct, utilize the quantity of spin adduct calculate original free radical number.This method not only contributes to verify the 26S Proteasome Structure and Function of biomacromolecule, and can provide various Useful Informations to the internal environment of various biosomes, space conformation and motion state.Vegetable cell has the structure that is different from zooblast, and by the selection of trapping agent, the optimization of contact conditions and extracting method, is expected to realize high sensitivity, hydroxyl free radical in plant body that antijamming capability is strong is measured.
Summary of the invention
The assay method that the object of this invention is to provide a kind of hydroxyl free radical in plant body.To achieve these goals, technical scheme of the present invention is to adopt electron spin resonance in conjunction with secondary free radical spin trapping technology, by a series ofly catching, extracting method, measures the hydroxy free radical concentration in plant.That this method has advantages of is simple to operate, highly sensitive, antijamming capability is strong.
The invention provides a kind of assay method of hydroxyl free radical in plant body, described determination method comprises the steps:
1) preparation of radical scavenger: take the N-tert-butyl group-α-benzene nitrone (purchased from: AMRESCO company), at room temperature be dissolved in dimethyl sulfoxide (DMSO) (purchased from: Sigma-Aldrich company), be mixed with dimethyl sulfoxide (DMSO) (DMSO) solution of the N-tert-butyl group-α-benzene nitrone (PBN) of 10-100mM;
2) (for example take fresh plant tissue, root, stem or leaf or its combination) 0.2-1.5g, shred and be put in glass tissue grinder, add step 1) in the dimethyl sulphoxide solution of the 0.5-1.5ml 10-100mM N-tert-butyl group-α-benzene nitrone of preparation, in ice bath, grind 1-5 minute to evenly;
3) by step 2) gained lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), the centrifugal 3-10 minute of 2000-6000g at 0-4 ℃; With
4) by step 3) sample of gained, get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: ESP 300, purchased from: Bruker company) detect.
According to the preferred embodiments of the invention, the assay method of described hydroxyl free radical in plant body comprises the steps:
1) preparation of radical scavenger: take the N-tert-butyl group-α-benzene nitrone (purchased from: AMRESCO company), at room temperature be dissolved in dimethyl sulfoxide (DMSO) (purchased from: Sigma-Aldrich company), be mixed with dimethyl sulfoxide (DMSO) (DMSO) solution of the N-tert-butyl group-α-benzene nitrone (PBN) of 10-100mM;
2) (for example take fresh plant tissue, root, stem or leaf or its combination) 0.2-1.5g, shred and be put in glass tissue grinder, add step 1) in the dimethyl sulphoxide solution of the 0.5-1.5ml 10-100mM N-tert-butyl group-α-benzene nitrone of preparation, in ice bath, grind 1-5 minute to evenly;
3) by step 2) gained lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), at 0-4 ℃ after the centrifugal 3-10 of 2000-6000g minute, abandoning supernatant;
4) by step 3) deposit sample of gained, the dimethyl sulphoxide solution that again adds the 0.5-1.5ml N-tert-butyl group-α-benzene nitrone, vibrate after 1-5 minute, in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge, the centrifugal 3-10 minute of 2000-6000g at 0-4 ℃; With
5) by step 4) sample of gained, get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: Bruker ESP 300, purchased from: Bruker company) detect.
Wherein, in one embodiment of the invention, above-mentioned steps 4) or 5) in Election spim resonance (model: the ESP 300 that mentions, purchased from: testing conditions Bruker company) detecting can be: microwave power 20mW, microwave frequency 9.7GHz, central magnetic field 3470G, field width 100G, frequency modulation 100kHz, amplitude modulation 2.5G.But those skilled in the art should understand that, the testing conditions that in actual experiment, selected Election spim resonance detects can be because of the model of Election spim resonance used, sample type etc. and different, and those skilled in the art can select according to physical condition or through simple preliminary experiment.
The dimethyl sulphoxide solution of the N-tert-butyl group-α-benzene nitrone using method of the present invention, described step 1) need measured preparation on the same day, preferably instant joining.
Method of the present invention, described plant is organized as plant roots, stem, leaf or its combination.
In addition, we are to above-mentioned steps 2)-4) in centrifugal rotational speed, centrifugation time be optimized, while finding that rotating speed is 2000g, sample separation weak effect, consequently spectrogram baseline is poor, a little less than Free Radical Signal, sensitivity is measured in impact relatively.Equally, when centrifugation time is 1 minute, sample separation weak effect, measures sensitivity not high.But if centrifugation time is long, the easy cancellation of free radical in sample, impact is measured.Therefore, in preferred embodiments, the centrifugal rotational speed that we preferably use is 4500g, and centrifugation time is 3-10 minute, and more preferably, centrifugation time is 4 minutes.
Temperature bathe be for the free radical addition reaction of the N-tert-butyl group-α-benzene nitrone faster.When temperature is 20 ℃, addition reaction is slow, a little less than causing measured signal; If but temperature is too high, radical life is short, easily cancellation, also can cause the reduction of sensitivity.Equally, the warm bath time is too short, and addition reaction is incomplete, and measured signal is low; But overlong time, the easy cancellation of free radical, also can cause measured signal to die down.Therefore, in preferred embodiments, the warm bath temperature that we preferably use is 37 ℃, and the warm bath time is 15 minutes.
Therefore,, in a preferred embodiment of the present invention, the assay method of described hydroxyl free radical in plant body comprises the steps:
1) preparation of radical scavenger: take the N-tert-butyl group-α-benzene nitrone (purchased from: AMRESCO company), at room temperature be dissolved in dimethyl sulfoxide (DMSO) (purchased from: Sigma-Aldrich company), be mixed with dimethyl sulfoxide (DMSO) (DMSO) solution of the N-tert-butyl group-α-benzene nitrone (PBN) of 10-100mM;
2) (for example take fresh plant tissue, root, stem or leaf or its combination) 0.2-1.5g, shred and be put in glass tissue grinder, add step 1) in the dimethyl sulphoxide solution of the 0.5-1.5ml 10-100mM N-tert-butyl group-α-benzene nitrone of preparation, in ice bath, grind 1-5 minute to evenly;
3) by step 2) gained lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 37 ℃ of water-baths, temperature is bathed 15 minutes, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), at 0-4 ℃, 4500g is after centrifugal 4 minutes, abandoning supernatant;
4) by step 3) deposit sample of gained, the dimethyl sulphoxide solution that again adds the 0.5-1.5ml N-tert-butyl group-α-benzene nitrone, vibrated after 1-5 minute, and in 37 ℃ of water-baths, temperature is bathed 15 minutes, then centrifuge tube is placed in to refrigerated centrifuge, at 0-4 ℃, 4500g is centrifugal 4 minutes; With
5) by step 4) sample of gained, get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: Bruker ESP 300, purchased from: Bruker company) detect.
The present invention adopts electron spin resonance in conjunction with secondary free radical spin trapping technology, by a series ofly catching, extracting method, measures the hydroxyl radical free radical in plant.Selected hydroxyl radical free radical trapping agent is dimethyl sulfoxide (DMSO) and the N-tert-butyl group-α-benzene nitrone, and its principle is to describe with following reaction equation:
DMSO+OH·→DMSO·OH+·CH
3
2·CH
3+O
2→2·OCH
3
·OCH
3+PBN→PBN/·OCH
3
·CH
3+PBN→PBN/·CH
3
PBN/OCH in formula
3and PBN/CH
3be more stable free radical, after testing, their life-span reaches 40 minutes.Using them as reporter group, carry out electron spin resonance detection, the ESR signal by means of this reporter group, quantitatively detects OH thereby reach.
By what report on document, with electron spin resonance, in conjunction with OH method in secondary free radical spin trapping technology mensuration animal body, be directly used in the OH measuring in plant, gained spectrogram baseline is poor, and a little less than Free Radical Signal, insufficient sensitivity is desirable.The inventor through selecting suitable to catch, extracting method, electron spin resonance spectroscopy figure obviously improves, and has greatly improved the sensitivity of method, can be used for the quantitative detection of hydroxyl free radical in plant body.This is advantage of the present invention place.
Determination method of the present invention is highly sensitive, and anti-plant base body interference performance is strong, can realize accurate, quick, the efficient detection of plant interior free yl, for pollutant causes the mechanism research of plant poisoning and the ecological toxicity early diagnosis of soil pollution, lays the foundation.
Accompanying drawing explanation
In detailed description below in conjunction with accompanying drawing, above-mentioned feature and advantage of the present invention will be more obvious, wherein:
Fig. 1. show the spin resonance spectrogram of hydroxyl radical free radical in the corn root under coercing of cadmium (Cd) in embodiment 1.
Fig. 2. in demonstration embodiment 2, Atrazine is coerced the spin resonance spectrogram of hydroxyl radical free radical in lower wheat root, leaf.
Fig. 3. in demonstration embodiment 3, variable concentrations aureomycin (CTC) is coerced the spin resonance spectrogram of hydroxyl radical free radical in lower corn root.
Fig. 4. show in embodiment 4 that difference is caught, under extraction conditions, the spin resonance spectrogram of hydroxyl radical free radical in corn root, a: temperature is bathed; B: through once temperature bath and centrifugal; C: through secondary temperature bath and centrifugal.
Fig. 5. show in embodiment 5 not under equality of temperature bath temperature condition the spin resonance spectrogram of hydroxyl radical free radical in corn root, a:20 ℃; B:37 ℃; C:45 ℃.
Fig. 6. in demonstration embodiment 6, equality of temperature is not bathed under time conditions, the spin resonance spectrogram of hydroxyl radical free radical in corn root, a:4 minute; B:15 minute; C:30 minute.
Fig. 7. show in embodiment 7 under different centrifuge speed conditions the spin resonance spectrogram of hydroxyl radical free radical in corn root, a:2000g; B:4500g; C:6000g.
Fig. 8. show under the different centrifugation time conditions of embodiment 8 the spin resonance spectrogram of hydroxyl radical free radical in corn root, a:1 minute; B:4 minute; C:10 minute.
Embodiment
Carry out by the following examples further to illustrate the present invention.But should be appreciated that, described embodiment is illustrational object, is not intended to limit scope and spirit of the present invention.
Embodiment 1: the hydroxyl radical free radical in the corn root of coercing in conjunction with secondary free radical spin trapping technology mensuration cadmium with electron spin resonance
Corn seedling root after germinateing 5 days is immersed in to 5mg/L Cd (NO
3)
2(purchased from: in aqueous solution Beijing chemical reagents corporation), after 24 hours, the root of separated seedling, leaf, clean root, leaf with tap water and distilled water respectively.Take fresh plant root 0.2-1.5g, shred and be put in glass tissue grinder, add the DMSO solution of 0.5-1.5ml 10-100mM PBN, in ice bath, grind evenly.By lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 20-45 ℃ of water-bath, temperature was bathed after 5-20 minute, centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), at 0-4 ℃, 2000-6000g refrigerated centrifuge is after 3-10 minute, abandoning supernatant.The DMSO solution that adds again 0.5-1.5ml PBN, vibration 1-5 minute, in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, 0-4 ℃, 2000-6000g refrigerated centrifuge 3-10 minute.Get supernatant and pack quartz glass samples pipe (model: 1mm into, purchased from: Bruker company), with Election spim resonance (model: ESP 300, purchased from: Bruker company) detect, testing conditions is as follows: microwave power 20mW, microwave frequency 9.7GHz, central magnetic field 3470G, field width 100G, frequency modulation 100kHz, amplitude modulation 2.5G.Detect spectrogram and see Fig. 1.
As can be seen from Fig. 1, there is obvious spike in spectrogram, and this peak is the peak (OCH of methoxy free radical
3).The generation of hydroxyl radical free radical in heavy metal cadmium energy inducing plant body is described.The method highly sensitive, antijamming capability is strong, can meet the qualitative determination of free radical in plant roots.
Embodiment 2: with electron spin resonance, in conjunction with secondary free radical spin trapping technology, measure Atrazine and coerce the hydroxyl radical free radical in lower wheat root, leaf
By the wheat seedling root after germinateing 5 days be immersed in 10mg/L Atrazine (purchased from: Sigma-Aldrich company).After 24 hours, the root of separated seedling, leaf, clean root, leaf with tap water and distilled water respectively.Take fresh plant root or leaf 0.2-1.5g, shred respectively and be put in glass tissue grinder, add the DMSO solution of 0.5-1.5ml10-100mM PBN, in ice bath, grind evenly.Lapping liquid is transferred in centrifuge tube, in 20-45 ℃ of water-bath, temperature was bathed after 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), at 0-4 ℃, 2000-6000g refrigerated centrifuge is after 3-10 minute, abandoning supernatant.The DMSO solution that adds again 0.5-1.5ml PBN, vibration 1-5 minute, in 20-45 ℃ of water-bath, temperature was bathed after 5-20 minute, in 0-4 ℃, 2000-6000g refrigerated centrifuge 3-10 minute.Get supernatant and pack quartz glass samples pipe into, with Election spim resonance, detect, testing conditions is as follows: microwave power 20mW, microwave frequency 9.7GHz, central magnetic field 3470G, field width 100G, frequency modulation 100kHz, amplitude modulation 2.5G.Detect spectrogram and see Fig. 2.
As can be seen from Fig. 2, there is obvious spike in spectrogram, and this peak is the free base peak (OCH of methoxy
3) and methyl free radicals peak (CH
3).The generation of hydroxyl radical free radical in Atrazine energy inducing plant body is described.The method highly sensitive, antijamming capability is strong, can meet the qualitative determination of free radical in plant roots, leaf.
Embodiment 3: the hydroxyl radical free radical in the corn root under coercing in conjunction with secondary free radical spin trapping technology mensuration variable concentrations aureomycin with electron spin resonance
Maize seedling root portion after germinateing 5 days is immersed in respectively in 0,0.5,5,50mg/L aureomycin (CTC, purchased from: Chinese medicine and biological products assay institute) aqueous solution.After 24 hours, the root of separated seedling, leaf, clean root with tap water and distilled water.Take fresh plant root 0.2-1.5g, shred and be put in glass tissue grinder, add the DMSO solution of 0.5-1.5ml 10-100mM PBN, in ice bath, grind evenly.Lapping liquid is transferred in centrifuge tube, in 20-45 ℃ of water-bath, temperature was bathed after 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), at 0-4 ℃, 2000-6000g refrigerated centrifuge is after 3-10 minute, abandoning supernatant.The DMSO solution that adds again 0.5-1.5ml PBN, vibration 1-5 minute, in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, 0-4 ℃, 2000-6000g refrigerated centrifuge 3-10 minute.Get supernatant and pack quartz glass samples pipe into, with Election spim resonance, detect, testing conditions is as follows: microwave power 20mW, microwave frequency 9.7GHz, central magnetic field 3470G, field width 100G, frequency modulation 100kHz, amplitude modulation 2.5G.Detect spectrogram and see Fig. 3.
As can be seen from Fig. 3, in the plant of normal growth, also hydroxyl radical free radical can be detected, the highly sensitive of the method is described.Along with the raising of chlortetracycline concentration, in plant roots, Free Radical Signal obviously improves, and has dose-effect relationship, illustrates that this assay method can meet the quantitative test of hydroxyl free radical in plant body.
Corn seedling root after germinateing 5 days is immersed in 50mg/L aureomycin (purchased from Chinese medicine and biological products assay institute) aqueous solution.After 24 hours, the root of separated seedling, leaf, use from meter Shui and distilled water root cleaned.Take respectively three parts of fresh plant root 0.2-1.5g, shred and be put in glass tissue grinder, add the DMSO solution of 0.5-1.5ml 10-100mM PBN, in ice bath, grind evenly.
The first duplicate samples is ground and to be transferred in centrifuge tube centrifugally, get supernatant and directly with Election spim resonance, detect that (spectrogram is shown in Fig. 4 a).
After the second duplicate samples is ground, be transferred in centrifuge tube, in 20-45 ℃ of water-bath, temperature was bathed after 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), at 0-4 ℃, 2000-6000g refrigerated centrifuge, after 3-10 minute, is got supernatant and directly with Election spim resonance, is detected (spectrogram is shown in Fig. 4 b).
After triplicate sample is ground, be transferred in centrifuge tube, in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), at 0-4 ℃, 2000-6000g refrigerated centrifuge is after 3-10 minute, abandoning supernatant.The DMSO solution that adds again 1-5ml PBN, vibration 1-5 minute, in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, 0-4 ℃, 2000-6000g refrigerated centrifuge 3-10 minute.Get supernatant and pack quartz glass samples pipe into, with Election spim resonance, detect (spectrogram is shown in Fig. 4 c).
As can be seen from Fig. 4, radical scavenger is added after sample and directly measured, gained spectrogram baseline is very poor, and a little less than Free Radical Signal, insufficient sensitivity is desirable.Through temperature once bathe and centrifugal after, spectrogram baseline, signal make moderate progress.Through secondary temperature bathe and centrifugal after, spectrogram quality obviously improves.
Embodiment 5: not under equality of temperature bath temperature condition, and the spin resonance spectrogram of hydroxyl radical free radical in corn root.
Corn seedling root after germinateing 5 days is immersed in to 5mg/L Cd (NO
3)
2(purchased from: in aqueous solution Beijing chemical reagents corporation), after 24 hours, the root of separated seedling, leaf, clean root, leaf with tap water and distilled water respectively.Take 3 parts of fresh plant root 0.2-1.5g, shred and be put in glass tissue grinder, add respectively the DMSO solution of 0.5-1.5ml 10-100mM PBN, in ice bath, grind evenly.
By the first duplicate samples lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 20 ℃ of water-baths, temperature is bathed 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), 2000-6000g refrigerated centrifuge 3-10 minute at 0-4 ℃, abandoning supernatant.The DMSO solution that adds again 0.5-1.5ml PBN, vibration 1-5 minute, in 20 ℃ of water-baths, temperature was bathed after 5-20 minute, in 0-4 ℃, 2000-6000g refrigerated centrifuge 3-10 minute.Get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: ESP 300, purchased from: Bruker company) detect.Detect spectrogram and see Fig. 5 a.
By the second duplicate samples lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 37 ℃ of water-baths, temperature is bathed 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), 2000-6000g refrigerated centrifuge 3-10 minute at 0-4 ℃, abandoning supernatant.The DMSO solution that adds respectively again 0.5-1.5ml PBN, vibration 1-5 minute, in 37 ℃ of water-baths, temperature is bathed 5-20 minute, 0-4 ℃, 2000-6000g refrigerated centrifuge 3-10 minute.Get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: ESP 300, purchased from: Bruker company) detect.Detect spectrogram and see Fig. 5 b.
By triplicate sample lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 45 ℃ of water-baths, temperature is bathed 5-20 minute, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), 2000-6000g refrigerated centrifuge 3-10 minute at 0-4 ℃, abandoning supernatant.The DMSO solution that adds respectively again 0.5-1.5ml PBN, vibration 1-5 minute, in 45 ℃ of water-baths, temperature is bathed 5-20 minute, 0-4 ℃, 2000-6000g refrigerated centrifuge 3-10 minute.Get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: ESP 300, purchased from: Bruker company) detect.Detect spectrogram and see Fig. 5 c.
As can be seen from Fig. 5, each is not under equality of temperature bath temperature, and obvious free base peak all appears in spectrogram, but 20 ℃, 45 ℃ temperature bathe sample peak heights and be significantly less than the peaks that 37 ℃ of temperature are bathed, and illustrates that too high and too low warm bath temperature all can affect the sensitivity of mensuration.Therefore, preferred heated culture temperature is 37 ℃.Embodiment 6: equality of temperature is not bathed under time conditions, the spin resonance spectrogram of hydroxyl radical free radical in corn root.
Corn seedling root after germinateing 5 days is immersed in to 5mg/L Cd (NO
3)
2(purchased from: in aqueous solution Beijing chemical reagents corporation), after 24 hours, the root of separated seedling, leaf, clean root, leaf with tap water and distilled water respectively.Take 3 parts of fresh plant root 0.2-1.5g, shred and be put in glass tissue grinder, add respectively the DMSO solution of 0.5-1.5ml 10-100mM PBN, in ice bath, grind evenly.
With above-mentioned three duplicate samples lapping liquids, by the step of embodiment 5, test respectively, different is that the present embodiment bath temperature control used is 37 ℃, and incubative time is respectively 4 minutes (the first duplicate samples), 15 minutes (the second duplicate samples) and 20 minutes (triplicate sample).The detection spectrogram of three duplicate samples lapping liquids is shown in Fig. 6.
As can be seen from Fig. 6, temperature bathe 4 minutes sample spectrogram (Fig. 6 a) in free base peak less obvious, in the sample spectrogram of temperature bath 15 minutes (Fig. 6 b) and 20 minutes (Fig. 6 c), there is obvious free base peak, the peak height of 20 minutes is less than 15 minutes, illustrates that the too short and long temperature time of bathing all can affect the sensitivity of mensuration.Therefore, preferred incubative time is 15 minutes.
Embodiment 7: under different centrifugal rotational speed conditions, and the spin resonance spectrogram of hydroxyl radical free radical in corn root.
Corn seedling root after germinateing 5 days is immersed in to 5mg/L Cd (NO
3)
2(purchased from: in aqueous solution Beijing chemical reagents corporation), after 24 hours, the root of separated seedling, leaf, clean root, leaf with tap water and distilled water respectively.Take 3 parts of fresh plant root 0.2-1.5g, shred and be put in glass tissue grinder, add respectively the DMSO solution of 0.5-1.5ml 10-100mM PBN, in ice bath, grind evenly.
By the first duplicate samples lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 37 ℃ of water-baths, temperature was bathed after 15 minutes, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), respectively at 2000g refrigerated centrifuge at 0-4 ℃ after 3-10 minute, abandoning supernatant.The DMSO solution that adds again 0.5-1.5ml PBN, vibration 1-5 minute, in 37 ℃ of water-baths, temperature is bathed after 15 minutes, in 0-4 ℃ of 2000g refrigerated centrifuge 3-10 minute.Get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: ESP 300, purchased from: Bruker company) detect.Detect spectrogram and see Fig. 7 a.
By the second duplicate samples lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 37 ℃ of water-baths, temperature was bathed after 15 minutes, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), at 0-4 ℃, 4500g refrigerated centrifuge is after 3-10 minute, abandoning supernatant.The DMSO solution that adds again 0.5-1.5ml PBN, vibration 1-5 minute, in 37 ℃ of water-baths, temperature was bathed after 15 minutes, in 0-4 ℃, 4500g refrigerated centrifuge 3-10 minute.Get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: ESP 300, purchased from: Bruker company) detect.Detect spectrogram and see Fig. 7 b.
By triplicate sample lapping liquid be transferred to the centrifuge tube that volume is 1.5ml (purchased from: Cang County, Hebei province Du Sheng Qiang Fa medical apparatus and instruments factory), in 37 ℃ of water-baths, temperature was bathed after 15 minutes, then centrifuge tube is placed in to refrigerated centrifuge (model: GL-8MS, purchased from: Shanghai Lu Xiang instrument hydro-extractor instrument company limited), respectively at 6000g refrigerated centrifuge at 0-4 ℃ after 3-10 minute, abandoning supernatant.The DMSO solution that adds again 0.5-1.5ml PBN, vibration 1-5 minute, in 37 ℃ of water-baths, temperature was bathed after 15 minutes, in 0-4 ℃, 6000g refrigerated centrifuge 3-10 minute.Get supernatant pack into quartz glass samples pipe (model: 1mm, purchased from: Bruker company), with Election spim resonance (model: ESP 300, purchased from: Bruker company) detect.Detect spectrogram and see Fig. 7 c.
As can be seen from Fig. 7, when centrifugal rotational speed is 2000g, spectrogram baseline is poor, and a little less than Free Radical Signal, sensitivity is measured in impact.Increase centrifugal rotational speed, sample separation is effective, occurs obvious free base peak in sample spectrogram, illustrates that slow centrifugal rotational speed can affect the sensitivity of mensuration.Therefore, preferred centrifugal rotational speed is 4500g.
Embodiment 8: under different centrifugation time conditions, and the spin resonance spectrogram of hydroxyl radical free radical in corn root.
Corn seedling root after germinateing 5 days is immersed in to 5mg/L Cd (NO
3)
2(purchased from: in aqueous solution Beijing chemical reagents corporation), after 24 hours, the root of separated seedling, leaf, clean root, leaf with tap water and distilled water respectively.Take 3 parts of fresh plant root 0.2-1.5g, shred and be put in glass tissue grinder, add the DMSO solution of 0.5-1.5ml 10-100mM PBN, in ice bath, grind evenly.
With above-mentioned three duplicate samples lapping liquids, by the step of embodiment 7, test respectively, different is that the present embodiment centrifugal speed used is 4500g, and centrifugation time is respectively 1 minute (the first duplicate samples), 4 minutes (the second duplicate samples) and 10 minutes (triplicate sample).The detection spectrogram of three duplicate samples lapping liquids is shown in Fig. 8.
As can be seen from Fig. 8, when centrifugation time is in the time of 1 minute, spectrogram baseline is poor, and a little less than Free Radical Signal, sensitivity is measured in impact.Increase centrifugal rotational speed, sample separation is effective, but centrifugal 10 minutes time in sample spectrogram free base peak low in the time of more centrifugal 4 minutes, illustrate that too short or long centrifugation time can affect the sensitivity of mensuration.Therefore, preferred centrifugation time is 3-10 minute, and preferred centrifugation time is 4 minutes.
Should be appreciated that, although exemplary embodiment shows particularly and describes the present invention, but will be understood by those skilled in the art that, under the condition not deviating from by the defined the spirit and scope of the present invention of accompanying claim, the variation of various forms and details can be carried out therein, the combination in any of various embodiments can be carried out.
Claims (10)
1. the assay method of a hydroxyl free radical in plant body, choose dimethyl sulfoxide (DMSO) and the N-tert-butyl group-α-benzene nitrone is made hydroxyl radical free radical trapping agent, through a series of catch, extract after, with electron spin paramagnetic resonance, measure radical adduct concentration in plant, described assay method comprises the steps:
1) preparation of radical scavenger: take the N-tert-butyl group-α-benzene nitrone, be at room temperature dissolved in dimethyl sulfoxide (DMSO), be mixed with the dimethyl sulphoxide solution of the N-tert-butyl group-α-benzene nitrone of 10-100mM;
2) take the 0.2-1.5g of fresh plant soma, shred and be put in glass tissue grinder, add step 1) in the dimethyl sulphoxide solution of the 0.5-1.5ml10-100mM N-tert-butyl group-α-benzene nitrone of preparation, in ice bath, grind 1-5 minute to evenly;
3) by step 2) gained lapping liquid is transferred in centrifuge tube, and in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, centrifuge tube is placed in to refrigerated centrifuge, the centrifugal 3-10 minute of 2000-6000g at 0-4 ℃; With
4) by step 3) sample of gained, get supernatant and pack quartz glass samples pipe into, with Election spim resonance, detect.
2. the assay method of a hydroxyl free radical in plant body, choose dimethyl sulfoxide (DMSO) and the N-tert-butyl group-α-benzene nitrone is made hydroxyl radical free radical trapping agent, through a series of catch, extract after, with electron spin paramagnetic resonance, measure radical adduct concentration in plant, described assay method comprises the steps:
1) preparation of radical scavenger: take the N-tert-butyl group-α-benzene nitrone, be at room temperature dissolved in dimethyl sulfoxide (DMSO), be mixed with the dimethyl sulphoxide solution of the N-tert-butyl group-α-benzene nitrone of 10-100mN4;
2) take the 0.2-1.5g of fresh plant soma, shred and be put in glass tissue grinder, add step 1) in the dimethyl sulphoxide solution of the 0.5-1.5ml10-100mM N-tert-butyl group-α-benzene nitrone of preparation, in ice bath, grind 1-5 minute to evenly;
3) by step 2) gained lapping liquid is transferred in centrifuge tube, and in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, and centrifuge tube is placed in to refrigerated centrifuge, at 0-4 ℃ after the centrifugal 3-10 of 2000-6000g minute, abandoning supernatant;
4) by step 3) sample of gained, the dimethyl sulphoxide solution that again adds the 0.5-1.5ml10-100mM N-tert-butyl group-α-benzene nitrone, vibrate after 1-5 minute, in 20-45 ℃ of water-bath, temperature is bathed 5-20 minute, centrifuge tube is placed in to refrigerated centrifuge instrument, the centrifugal 3-10 minute of 2000-6000g at 0-4 ℃; With
5) by step 4) sample of gained, get supernatant and pack quartz glass samples pipe into, with Election spim resonance, detect.
3. the assay method of hydroxyl free radical in plant body according to claim 1 and 2, is characterized in that, described plant is organized as the root of plant, stem, leaf or its combination.
4. the assay method of hydroxyl free radical in plant body according to claim 1 and 2, is characterized in that, the warm bath temperature of described water-bath is 37 ℃.
5. the assay method of hydroxyl free radical in plant body according to claim 4, is characterized in that, in described water-bath, temperature is bathed 15 minutes.
6. the assay method of hydroxyl free radical in plant body according to claim 1 and 2, is characterized in that, in described water-bath, temperature is bathed 15 minutes.
7. according to the assay method of the hydroxyl free radical in plant body described in any one in claim 1,2 or 5, it is characterized in that, described centrifugal rotating speed is 4500g.
8. according to the assay method of the hydroxyl free radical in plant body described in any one in claim 1,2 or 5, it is characterized in that, the described centrifugal time is 4 minutes.
9. the assay method of hydroxyl free radical in plant body according to claim 7, is characterized in that, the described centrifugal time is 4 minutes.
10. the assay method of hydroxyl free radical in plant body according to claim 4, is characterized in that, the described centrifugal time is 4 minutes.
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| EP1210299B1 (en) * | 1999-09-01 | 2004-03-10 | University of Abertay Dundee | Method of producing hydroxyl radicals for chemical reactions |
| CN101413896A (en) * | 2008-12-02 | 2009-04-22 | 上海理工大学 | Method for measuring hydroxy free radical |
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