CN109164079A - Aluminum ions detection method in a kind of plant tissue - Google Patents
Aluminum ions detection method in a kind of plant tissue Download PDFInfo
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- CN109164079A CN109164079A CN201811103361.3A CN201811103361A CN109164079A CN 109164079 A CN109164079 A CN 109164079A CN 201811103361 A CN201811103361 A CN 201811103361A CN 109164079 A CN109164079 A CN 109164079A
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- 238000001514 detection method Methods 0.000 title claims abstract description 43
- 229910052782 aluminium Inorganic materials 0.000 title claims abstract description 31
- -1 Aluminum ions Chemical class 0.000 title claims abstract description 23
- 238000004043 dyeing Methods 0.000 claims abstract description 33
- 150000001875 compounds Chemical class 0.000 claims abstract description 20
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical compound C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000001035 drying Methods 0.000 claims abstract description 11
- 239000003960 organic solvent Substances 0.000 claims abstract description 8
- 238000004090 dissolution Methods 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 238000007710 freezing Methods 0.000 claims abstract description 4
- 230000008014 freezing Effects 0.000 claims abstract description 4
- 125000001038 naphthoyl group Chemical group C1(=CC=CC2=CC=CC=C12)C(=O)* 0.000 claims description 19
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 238000010791 quenching Methods 0.000 claims description 11
- 230000000171 quenching effect Effects 0.000 claims description 11
- 230000005284 excitation Effects 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 239000011521 glass Substances 0.000 claims description 7
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-UHFFFAOYSA-N 0.000 claims description 6
- ZHBOFZNNPZNWGB-UHFFFAOYSA-N 9,10-bis(phenylethynyl)anthracene Chemical compound C1=CC=CC=C1C#CC(C1=CC=CC=C11)=C(C=CC=C2)C2=C1C#CC1=CC=CC=C1 ZHBOFZNNPZNWGB-UHFFFAOYSA-N 0.000 claims description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 238000013019 agitation Methods 0.000 claims description 3
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 claims description 3
- 239000012043 crude product Substances 0.000 claims description 3
- 235000019441 ethanol Nutrition 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- ATHHXGZTWNVVOU-UHFFFAOYSA-N N-methylformamide Chemical compound CNC=O ATHHXGZTWNVVOU-UHFFFAOYSA-N 0.000 claims 2
- REDXJYDRNCIFBQ-UHFFFAOYSA-N aluminium(3+) Chemical compound [Al+3] REDXJYDRNCIFBQ-UHFFFAOYSA-N 0.000 abstract description 36
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 241000196324 Embryophyta Species 0.000 description 54
- 239000000243 solution Substances 0.000 description 13
- YXOLAZRVSSWPPT-UHFFFAOYSA-N Morin Chemical compound OC1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 YXOLAZRVSSWPPT-UHFFFAOYSA-N 0.000 description 11
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 11
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 description 11
- 235000007708 morin Nutrition 0.000 description 11
- 239000000975 dye Substances 0.000 description 10
- 239000004411 aluminium Substances 0.000 description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical group CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000012545 processing Methods 0.000 description 5
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 241000208340 Araliaceae Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 238000001917 fluorescence detection Methods 0.000 description 2
- 235000008434 ginseng Nutrition 0.000 description 2
- 230000001744 histochemical effect Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007447 staining method Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000790917 Dioxys <bee> Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- ZOKXTWBITQBERF-UHFFFAOYSA-N Molybdenum Chemical compound [Mo] ZOKXTWBITQBERF-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229960001760 dimethyl sulfoxide Drugs 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- AWJUIBRHMBBTKR-UHFFFAOYSA-N isoquinoline Chemical compound C1=NC=CC2=CC=CC=C21 AWJUIBRHMBBTKR-UHFFFAOYSA-N 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 229910052750 molybdenum Inorganic materials 0.000 description 1
- 239000011733 molybdenum Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
- GPPXJZIENCGNKB-UHFFFAOYSA-N vanadium Chemical compound [V]#[V] GPPXJZIENCGNKB-UHFFFAOYSA-N 0.000 description 1
- 239000005418 vegetable material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
- G01N2001/2873—Cutting or cleaving
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The present invention relates to aluminum ions detection method in a kind of plant tissue, which includes the following steps: that (a) obtains dyeing liquor using organic solvent dissolution naphthalimide based compound；(b) by plant sample freezing, slice, preservation, plant section is obtained；(c) by step (b) plant section drying, then be placed in step (a) in dyeing liquor in be protected from light dyeing, after dyeing, taking-up plant section washed, mounting；(d) plant section that step (c) obtains is detected using fluorescence microscope；The detection method can aluminium ion in detection plant accurate, efficiently, easy, and can determine distributing position of the aluminium ion in plant tissue cell, and aluminum ions relative amount is calculated by fluorescence intensity；In addition, high using the detection method high specificity, sensitivity and clarity.
Description
Technical field
The present invention relates to fluorescence detection fields, in particular to aluminum ions detection method in a kind of plant tissue.
Background technique
Aluminium (Aluminum, Al) is the most abundant metallic element of content in the earth's crust, and aluminium can be in the acid soil of pH < 5
Al3+Form, which is released, generates toxic action to plant, influences crop growth.As global soil acidizing degree rises,
50% cultivable soil is all acid soil.Therefore, aluminium poison has become a key constraints of world crops yield.
In the research of plant aluminum ionic stress, the detection method of aluminium is to evaluate an important finger of aluminium toxicity in plant
Mark, to the Aluminum toxicity of vegetable material carry out it is reliable and accurately identify be it is highly important, this is subsequent development scientific research work
The basic guarantee of work.Currently used method includes histochemical staining method, spectrophotometry, atomic absorption spectrography (AAS) and inductance
Coupling plasma method.Traditional histochemical staining method often uses morin (morin) as dyestuff, can be with aluminium ion knot
It closes, is formed with the compound that can produce fluorescence, can judge aluminium ion accumulation in cell further according to complex fluorescence intensity
Degree, but since fluorescent dye morin can be combined with aluminium, calcium, molybdenum, vanadium and iron, the specificity combined with aluminium ion is
It is adjusted by specific buffer environment, but portion is it is difficult to ensure that its stable buffer environment, leads to morin in the cell
It dyes to aluminum ions specificity, dye levels, sensitivity is lower and belongs to unspecific staining.
In view of this, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide aluminum ions detection method in a kind of plant tissue, which can be quasi-
Really, the aluminium ion in detection plant efficiently, easy, and can determine distributing position of the aluminium ion in plant tissue cell,
And aluminum ions relative amount is calculated by fluorescence intensity；In addition, using the detection method high specificity, sensitivity and
Clarity is high.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention provides aluminum ions detection method in a kind of plant tissue, which includes the following steps:
(a) dyeing liquor is obtained using organic solvent dissolution naphthalimide based compound；
(b) by plant sample freezing, slice, preservation, plant section is obtained；
(c) it is protected from light dyeing in the dyeing liquor dry the plant section in step (b), being then placed in step (a), is dyed
After, take out plant section washed, mounting；
(d) plant section that step (c) obtains is detected using fluorescence microscope.
By above-mentioned detection method can aluminium ion in detection plant accurate, efficiently, easy, and can determine aluminium from
Distributing position of the son in plant tissue cell, and aluminum ions relative amount is calculated by fluorescence intensity；In addition, adopting
It is high with the detection method high specificity, sensitivity and clarity.
Further, it includes being calculated by image analysis software that the fluorescence microscope, which carries out detection to plant section,
Fluorescence intensity；Preferably, described image analysis software is IMAGE-PRO PREMIER or CF-2000.
Naphthoyl imino-compound structural formula is in the present inventionPreparation method includes following step
It is rapid: by BPEA ([4- (- 1 hydrogen of 2- butyl -1-1,3- dioxy -2,3- dihydro-phenylpropyl alcohol isoquinolin -6)-piperazidine] acetic acid second
Ester) and 2- ethylaminoethanol be dissolved in acetonitrile, under agitation, flow back 13-15h, after being cooled to room temperature be concentrated in vacuo, purify
Obtain above-mentioned naphthoyl imino-compound；
Preferably, the mass ratio of the BPEA and 2- ethylaminoethanol is (4-5): 1；
Preferably, the purifying includes: using volume ratio for 1: the methanol-chloroform solution of (18-22) is as eluent to true
The crude product that sky is concentrated to get is eluted；Eluent is recrystallized by ethyl alcohol again.
By the selection of above-mentioned specific naphthalimide based compound, the naphthoyl imino-compound can with aluminium in plant from
Son specific binding, can be improved the accuracy of detection；In addition, by being sliced to plant sample, drying and processing, same energy
Enough promote the compound to enter plant cell sufficiently to be combined with the aluminium ion in sample, and can be improved the light transmission of plant tissue
Rate is conducive to fluorescence microscope, improves clarity；In addition particular dye (the i.e. naphthalene can be slowed down by carrying out mounting processing to slice
Imide compound) fluorescent quenching, improve detection accuracy.
Organic solvent is not limited strictly in the present invention, it is preferable that the organic solvent is selected from dimethylformamide, two
Any one in methyl sulfoxide and methanol.By the selection of specific organic solvent in the present invention, the compound can be dissolved
Except, additionally it is possible to plant sample is fixed, the fixation to plant tissue is completed while dyeing, improves plant sample group
The stability for knitting structure reduces influence of the fixation procedure to aluminium ion concentration in plant, and promotes the compound to plant
The infiltration of sample improves the joint efficiency of aluminium ion and the compound in sample.
Further, naphthoyl imino-compound concentration is 10 in dyeing liquor of the present invention-4-10-5mol/L；It is preferred that dense
Degree is 10-4mol/L；By the restriction to naphthoyl imino-compound concentration, naphthoyl imino-compound can be promoted to enter plant
Object tissue, and sufficiently the accuracy of detection is improved with combination aluminum ions in plant tissue.
Further, further include in the step (b)；Before slice, using Tissue-Tek frozen embedding agent to plant
Sample is embedded；Further, before preservation, by slice sticker on anticreep glass slide.
Further, it in the step (b), freezes as liquid nitrogen frozen；Slice thickness is 5-20 μm；Storage temperature is ﹣ 80
70 DEG C of~﹣.By above-mentioned specific processing and the limitation of parameter, the stability of plant sample can be preferably improved, is improved glimmering
The clarity of light；Preferably, plant sample is cleaned before freezing, and is blotted using filter paper.
Further, in the step (c), drying temperature is 50-60 DEG C, drying time 1.5-2.5min；It is protected from light anti-
It is 50-130min between seasonable.By the limitation of above-mentioned temperature, it can be avoided plant sample tissue and cause to damage during the drying process
Wound, can promote the abundant combination of aluminium ion and dyestuff in plant sample by the limitation in reaction time, improve aluminum ions inspection
Survey accuracy.
Preferably, in the step (c), mounting is carried out using anti-fluorescent quenching mountant；It is highly preferred that the anti-fluorescence
Mountant is quenched and is selected from Fluoromount-GTMAnti- fluorescent quenching mountant,Mountant and
Any one in the anti-fluorescent quenching mountant of Beyotime.By the specific selection of mountant, the particular dye can be slowed down
Fluorescent quenching, conducive to improve aluminium ion detection accuracy.
Fluorescence microscope is not limited strictly in the present invention, it is preferable that the fluorescence microscope is that common fluorescent is micro-
Mirror or laser confocal fluorescence microscope.
Further, the excitation wavelength of the laser confocal fluorescence microscope is 400-410nm, launch wavelength 500-
750nm, it is preferable that the excitation wavelength of the laser confocal fluorescence microscope is 405nm, launch wavelength 530nm；It is described general
Logical fluorescence microscope selects ultraviolet filter, and excitation wavelength is 350-400nm, it is preferable that the common fluorescent microscope
Ultraviolet filter is selected, and excitation wavelength is 350nm.By adjusting excitation wavelength for different fluorescence microscopes in the present invention
It can be realized and fluorescence detection is carried out to the dyestuff specifically bound with aluminium ion；Wherein, laser confocal fluorescence microscope passes through
Select the launch wavelength range (500-750nm) of acquisition, additionally it is possible to plant sample autofluorescence (launch wavelength < 488nm) be discharged
Interference.
Compared with prior art, beneficial effects of the present invention include at least:
(1) aluminium ion in the detection plant that detection method of the present invention can be accurate, efficient, easy, and can be true
Distribution situation and aluminum ions relative amount of the aluminium ion in plant tissue are determined, in addition, using detection method specificity
By force, sensitivity and clarity are high.
(2) plant sample can be fixed in the present invention by the selection of specific organic solvent, improves plant sample
The stability of this institutional framework, and promote infiltration of the naphthalimide based compound to plant sample, improve aluminium ion in sample
With the joint efficiency of naphthalimide based compound.
(3) present invention can be improved the specificity and sensitivity of aluminium ion dyeing, be conducive to by the selection of particular dye
To positioning aluminum ions in cell and relative quantification observation.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the fluorogram of sample longitudinal section after aluminium ion is handled 0 hour in first group in the application experimental example 2；
Fig. 2 is the fluorogram of sample slice after aluminium ion is handled 24 hours in first group in the application experimental example 2；
Fig. 3 is the fluorogram of sample transverse section after aluminium ion is handled 48 hours in first group in the application experimental example 2；
Fig. 4 is the fluorogram of sample longitudinal section after aluminium ion is handled 48 hours in first group in the application experimental example 2；
Fig. 5 is the fluorogram of sample slice after aluminium ion is handled 0 hour in second group in the application experimental example 2；
Fig. 6 is the fluorogram of sample slice after aluminium ion is handled 24 hours in second group in the application experimental example 2；
Fig. 7 is the fluorogram of sample transverse section after aluminium ion is handled 48 hours in second group in the application experimental example 2；
Fig. 8 is the fluorogram of sample longitudinal section after aluminium ion is handled 48 hours in second group in the application experimental example 2.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products obtained can be bought by city.
Naphthoyl imino-compound is prepared via a method which to obtain in each embodiment:
BPEA and 2- ethylaminoethanol is dissolved in acetonitrile, under agitation, flow back 14h, is cooled to room temperature rear vacuum
Concentration；It is eluted again using the methanol-chloroform solution that volume ratio is 1: 20 as eluent to crude product is concentrated in vacuo and obtained；
Eluent is recrystallized by ethyl alcohol again；
Wherein, the mass ratio of BPEA and 2- ethylaminoethanol is 5:1.
Embodiment 1
The present embodiment is aluminum ions detection method in a kind of plant tissue, which includes the following steps:
(a) dyeing liquor is obtained with dimethylformamide dissolution naphthalimide based compound, wherein naphthoyl in the dyeing liquor
Imino-compound concentration is 10-5mol/L；
(b) plant sample wash and be then rapidly frozen using liquid nitrogen using filter paper suck dry moisture, used
Tissue-Tek frozen embedding agent is embedded, then the sample after embedding is sliced, and slice thickness is 5 μm, by slice sticker
It is saved on anticreep glass slide in the case where 80 DEG C of ﹣；
(c) slice in step (b) is dried under the conditions of 50 DEG C 2.5min, then the slice after drying is placed in step
(a) it is protected from light 50min in the dyeing liquor in, then, takes out slice and is washed, then use Fluoromount-GTMAnti- fluorescence
Mountant is quenched and carries out mounting；
(d) slice that step (c) obtains is observed using laser confocal fluorescence microscope, then, and passes through figure
As fluorescence intensity is calculated in analysis software (CF-2000), wherein the excitation wavelength of laser confocal fluorescence microscope is
405nm, launch wavelength 530nm.
Embodiment 2
The present embodiment is aluminum ions detection method in a kind of plant tissue, which includes the following steps:
(a) dyeing liquor is obtained with dmso solution naphthalimide based compound, wherein naphthoyl is sub- in the dyeing liquor
Amino-compound concentration is 10-4mol/L；
(b) plant sample wash and be then rapidly frozen using liquid nitrogen using filter paper suck dry moisture, used
Tissue-Tek frozen embedding agent is embedded, then the sample after embedding is sliced, and slice thickness is 20 μm, by slice sticker
It is saved on anticreep glass slide in the case where 70 DEG C of ﹣；
(c) slice in step (b) is dried under the conditions of 60 DEG C 1.5min, then the slice after drying is placed in step
(a) it is protected from light 130min in the dyeing liquor in, then, takes out slice and is washed, then use
Mountant carries out mounting；
(d) slice that step (c) obtains is observed using laser confocal fluorescence microscope, then, and passes through figure
As fluorescence intensity is calculated in analysis software (IMAGE-PRO PREMIER), wherein the excitation of laser confocal fluorescence microscope
Wavelength is 400nm, launch wavelength 550nm.
Embodiment 3
The present embodiment is aluminum ions detection method in a kind of plant tissue, which includes the following steps:
(a) dyeing liquor is obtained with methanol dissolution naphthalimide based compound, wherein naphthoyl imino group in the dyeing liquor
Compound concentration is 5 × 10-5mol/L；
(b) plant sample wash and be then rapidly frozen using liquid nitrogen using filter paper suck dry moisture, used
Tissue-Tek frozen embedding agent is embedded, then the sample after embedding is sliced, and slice thickness is 13 μm, by slice sticker
It is saved on anticreep glass slide in the case where 70 DEG C of ﹣；
(c) slice in step (b) is dried under the conditions of 55 DEG C 2min, then the slice after drying is placed in step (a)
In dyeing liquor in be protected from light 70min, then, take out slice washed, then use the anti-fluorescent quenching mounting of Beyotime
Agent carries out mounting；
(d) slice that step (c) obtains is observed using common fluorescent microscope, and passes through image analysis software
Fluorescence intensity is calculated in (IMAGE-PRO PREMIER), wherein common fluorescent microscope selects ultraviolet filter, and swashs
Hair wavelength is 400nm.
Embodiment 4
The present embodiment is aluminum ions detection method in a kind of plant tissue, which includes the following steps:
(a) dyeing liquor is obtained with dmso solution naphthalimide based compound, wherein naphthoyl is sub- in the dyeing liquor
The compound concentration of amino is 10-4mol/L；
(b) plant sample wash and be then rapidly frozen using liquid nitrogen using filter paper suck dry moisture, used
Tissue-Tek frozen embedding agent is embedded, then the sample after embedding is sliced, and slice thickness is 10 μm, by slice sticker
It is saved on anticreep glass slide in the case where 80 DEG C of ﹣；
(c) slice in step (b) is dried under the conditions of 60 DEG C 2min, then the slice after drying is placed in step (a)
In dyeing liquor in be protected from light 100min, then, take out slice washed, then use the anti-fluorescence of Fluoromount-GTM
Mountant is quenched and carries out mounting；
(d) slice that step (c) obtains is observed using common fluorescent microscope, then, and passes through image analysis
Fluorescence intensity is calculated in software (CF-2000), wherein common fluorescent microscope selects ultraviolet filter, and excitation wavelength
For 350nm.
Reference examples 1
This reference examples is aluminum ions detection method in a kind of plant tissue, the detection side of the detection method and embodiment 4
Method is essentially identical, and difference, which is only that, is substituted for morin for naphthalimide based compound, and the concentration of morin is 10-3mol/
L。
Experimental example 1
Dyeing liquor is obtained using dmso solution naphthalimide based compound, wherein naphthoyl is sub- in the dyeing liquor
The concentration of the compound of amino is 10-4mol/L；
It is respectively configured containing Fe3+、Ca2+、Mg2+、Zn2+Mixed solution and AL3+Solution；Its each ion concentration is identical；
Equivalent dyeing liquor is added into mixed solution and aluminium ion solution respectively is protected from light 60min；
It detects the fluorescence intensity for being protected from light rear mixed liquor and aluminium ion solution respectively using sepectrophotofluorometer, examines
Surveying result is that mixed liquor does not find fluorescence phenomenon, and aluminium ion solution has fluorescence phenomenon, thus illustrates naphthoyl imino
Closing object has good specificity to aluminium ion.
Experimental example 2
One, material and reagent
Material: by aluminium ion Stress treatment 0h, for 24 hours with plant after 48h (ginseng) root system；
Reagent: Fluoromount-GTMAnti- fluorescent quenching mountant, the agent of Tissue-Tek frozen embedding, glass slide；
Two, detection method
Aluminium ion is handled 0h, wash with plant (ginseng) root system after 48h for 24 hours and blots water using filter paper respectively
Point, then, by the root system of plant handled well be divided into two groups, every group comprising aluminium ion processing 0h, for 24 hours with the plant roots of 48h
System:
First group of root system of plant is detected using the detection method of embodiment 4, is taken pictures, fluorescence intensity is taken pictures referring to table 1
As a result referring to figures 1-4；
Second group of root system of plant is detected using the detection method of reference examples 1, is taken pictures, fluorescence intensity is taken pictures referring to table 1
As a result referring to figure 5-8；
Table 1
As shown in Table 1
When aluminium ion handles 0h, it is glimmering that naphthoyl imino-compound fluorescent staining intensity is significantly smaller than morin dyeing dyeing
Luminous intensity can dye morin there are false positive phenomenon, and morin dyeing accuracy and specificity are poor.
The extension of time is handled according to aluminium ion, fluorescence intensity is in rising trend, illustrates that naphthoyl imino-compound dyes
Fluorescence intensity is positively correlated with the aluminium ion processing time.
In the case where naphthoyl imino-compound an order of magnitude lower than morin concentration, naphthoyl imino-compound
Fluorescent staining intensity is higher than the fluorescent staining intensity of morin, it is seen that the sensitivity of naphthoyl imino-compound is higher.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (10)
1. aluminum ions detection method in a kind of plant tissue, which comprises the steps of:
(a) dyeing liquor is obtained using organic solvent dissolution naphthalimide based compound；
(b) by plant sample freezing, slice, preservation, plant section is obtained；
(c) it is protected from light dyeing in the dyeing liquor dry the plant section in step (b), being then placed in step (a), dyeing terminates
Afterwards, take out plant section washed, mounting；
(d) plant section that step (c) obtains is detected using fluorescence microscope.
2. detection method according to claim 1, which is characterized in that the preparation method packet of the naphthoyl imino-compound
It includes: BPEA and 2- ethylaminoethanol is dissolved in acetonitrile, under agitation, flow back 13-15h, and it is dense to be cooled to room temperature rear vacuum
Contracting, purifying obtain above-mentioned naphthoyl imino-compound；
Preferably, the mass ratio of the BPEA and 2- ethylaminoethanol is (4-5): 1；
Preferably, the purifying includes: using volume ratio for 1: the methanol-chloroform solution of (18-22) is dense to vacuum as eluent
The obtained crude product that contracts is eluted；Eluent is recrystallized by ethyl alcohol again.
3. detection method according to claim 1, which is characterized in that in the step (a), the organic solvent is selected from two
Any one in methylformamide, dimethyl sulfoxide and methanol.
4. detection method according to claim 1, which is characterized in that naphthoyl imino-compound concentration in the dyeing liquor
It is 10-4-10-5mol/L。
5. detection method according to claim 1, which is characterized in that in the step (b), freeze as liquid nitrogen frozen；It cuts
Piece is with a thickness of 5-20 μm；Storage temperature is 70 DEG C of 80~﹣ of ﹣.
6. detection method according to claim 1, which is characterized in that further include in the step (b)；Before slice, use
Tissue-Tek frozen embedding agent embeds plant sample；
Preferably, before preservation, by slice sticker on anticreep glass slide.
7. detection method according to claim 1, which is characterized in that in the step (c), drying temperature is 50-60 DEG C,
Drying time is 1.5-2.5min；Being protected from light the time is 50-130min.
8. detection method according to claim 1, which is characterized in that in the step (c), mounting uses anti-fluorescent quenching
Mountant carries out；
Preferably, the anti-fluorescent quenching mountant is selected from Fluoromount-GTMAnti- fluorescent quenching mountant,Any one in the anti-fluorescent quenching mountant of mountant, Beyotime.
9. detection method according to claim 1, which is characterized in that the fluorescence microscope be common fluorescent microscope or
Laser confocal fluorescence microscope.
10. detection method according to claim 9, which is characterized in that the excitation of the laser confocal fluorescence microscope
Wavelength is 400-410nm, launch wavelength 500-750nm；
Preferably, the microscopical excitation wavelength of the common fluorescent is 350-400nm.
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