CN103302307A - Preparation method of silver nano particles and method for facilitating germination of cucumber seeds and growth and development of seedlings with silver nano particles - Google Patents
Preparation method of silver nano particles and method for facilitating germination of cucumber seeds and growth and development of seedlings with silver nano particles Download PDFInfo
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Abstract
The invention belongs to the technical field of nano material preparation and performance study, and discloses a preparation method of silver nano particles and a method for facilitating germination of cucumber seeds and growth and development of seedlings with the silver nano particles. The preparation method comprises the following steps that an AgNO3 aqueous solution is taken; PVP (Polyvinyl Pyrrolidone) is added; an amino acid aqueous solution which is isometric with the AgNO3 aqueous solution and has reducibility is added after PVP is dissolved completely; when a color of a reaction system graduates into brown and is optically transparent, the nano particles are generated and are smaller in dimension; centrifugal treatment is performed; an obtained supernatant is recollected into an initial reaction container; a precipitate obtained at the bottom of a centrifugal tube is stored in deionized water; and an Ag nano particle solution is obtained. The silver nano particles are prepared by taking amino acid as a reducing agent, so that the silver nano particles have good biocompatibility. PVP is taken as a dispersing agent for preventing aggregation of the nano particles. A requirement of the whole preparation on reaction conditions is not high; scale production is easy to realize; and the provided nano particles can be applied in agricultural production.
Description
Technical field
The invention belongs to nano material preparation and performance study technical field, i.e. the method for the preparation method of Nano silver grain and promotion germination of cucumber seeds thereof, seedling development.
Background technology
Between recent two decades in the past, nano science and nanometer technology have been subject to paying close attention to widely and having obtained development at full speed.In various nano materials, Nano silver grain is a study hotspot always.This is because Nano silver grain not only has the character such as small-size effect, skin effect, quantum size effect and macro quanta tunnel effect that general nano particle has, a member as noble metal, it has unique light, electricity, catalytic property, can be widely used as catalyst, battery electrode, low temperature heat conduction and conductive material etc.In addition, it is exactly to have powerful bactericidal effect that Nano silver grain also has a very concerned characteristic, common antibiotic is general a kind of can only kill 6 kinds of pathogen, and Nano silver grain can kill 650 various bacteria in several minutes, this is because Nano silver grain can directly enter thalline and be combined with the oxygen metabolism enzyme, thalline is choked to death, and the also mechanism of action of a uniqueness Just because of this is so that Nano silver grain has had the anti-microbial property of wide spectrum.And because Nano Silver belongs to the non-antibiotic bactericide, so that bacterium can't produce drug resistance, can effectively avoid the repeatedly outbreak obstinate that causes because of drug resistance.And the security of Nano silver grain is that international medical community is generally acknowledged, because the trace silver element was exactly one of necessary important element of human body originally, the nano-silver ionic neutral, can in human body, not be combined and deposit by various bioactivators, absorption and kill bacteria in pore, and can in body, discharge fully, can not produce toxic and side effect.Put down in writing in the Compendium of Material Medica of China: give birth to silver, nontoxic.Explanation in U.S. public health bureau nineteen ninety " about the survey report of silver-colored toxicity ": silver to human body without obvious toxic-side effects; Nano Silver is local application, and silver content is few, is safest application method.Investigate to find mouse at oral maximal tolerance dose 925mg/kg through test, when namely being equivalent to 4625 times of clinical using dosage, without any toxic reaction, in the skin irritation test of rabbit, also do not find any IR.In addition, Nano silver grain can also promote the healing of wound, the growth of cell and the reparation of damaged cell.Therefore Nano Silver is the natural antibacterial agent of latest generation, various Nano Silver products have penetrated into the every aspect of people's life, such as water purification filter material, antibiotic paint, series of amenities, anti-bacterial fibre cloth series, all kinds of cleaning agent series, novel antibacterial application series etc.
Studies show that in recent years, nano material not only can exert an influence to the vital movement of animal (comprising the mankind), and the physiological activity of plant is also had obvious effect.Nano material has begun to use at fresh-keeping, fertilizer synergistic, Animal Genetics and the feed development etc. of agriculture field.For example, the researcher of U.S. University of Arkansas is exposed to tamato seed under the CNT environment, found that, these germination speed are more fast again than common seed, and the seedling weight that has just sprouted is also heavy a lot of than common seedling.They think that CNT can " penetrate " the hard shell of seed, thereby greatly improve the water absorbing capacity of seed.For another example, there are some researches show that nano titanium oxide can promote to hide the sprouting of oriental wormwood and rape seed, CNT promotes the sprouting of soya seeds and growth etc. under can also drought stress.
Although people have poured into a large amount of energy on the performance study of Nano silver grain, substantially all rest on device preparation and antibacterial applications field, on the situation that affects of plant and the mechanism of action but nobody study.In addition, the preparation process that is applied to the Nano silver grain of biological field also must be biocompatibility, to avoid owing to the application that noxious material has affected Nano silver grain is introduced in preparation.
The present invention proposes a kind of green, the nontoxic method for preparing the biocompatibility Nano silver grain, the method is hanged down consumption environment protection, and the gained nano-particles size is even, is easy to realize batch production.After the Nano silver grain of this method gained processed the seed (the model seed is cucumber) of plant, seed germination speed was apparently higher than control group, and seedling is also obviously sturdy than control group.Although the more existing researchs of the application of nanometer technology in the crop seed soaking, relevant silver nanoparticle there is not yet relevant report to the impact of germination of cucumber seeds.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of green, the Nano silver grain that nontoxic, preparation size is even, biocompatibility is good.
Another object of the present invention provides a kind of method of utilizing these Nano silver grains to promote germination of cucumber seeds, seedling development.
The solution of the present invention is: a kind of preparation method of Nano silver grain is characterized in that step is as follows:
Get AgNO
3The aqueous solution adds polyvinylpyrrolidone, dissolves rear adding and AgNO fully until polyvinylpyrrolidone
3The isopyknic amino acid solution with reproducibility of the aqueous solution, when the color of reaction system gradually becomes brown and optical clear, nano particle generated and size less, centrifugal treating, the gained supernatant liquor is collected back the initial reaction container again, the precipitation that obtains in the centrifuge tube bottom is stored in deionized water, obtains the Ag nano-particle solution.
Specifically, the preparation method of nano particle is characterized in that step is as follows:
(1) under temperature 15-25 ℃ of condition, configuration concentration is 0.05-0.2 molL respectively
-1AgNO
3The aqueous solution and concentration 0.05-0.2 molL
-1The amino acid solution with reproducibility.The Ag nano particle that this concentration range prepares is at 20-30 nm, size uniform, decentralization is better, concentration lower or higher all can so that the size range of nano particle enlarge, even not.Although also have other reducing agents, can use such as sodium borohydride, hydrazine hydrate etc., these reducing agents all in various degree have toxicity so that the biocompatibility of nano particle reduces.Amino acid with reproducibility is preferably α-aminopropionic acid.And α-aminopropionic acid is a seed amino acid, itself is exactly biocompatible, and the good biocompatibility that the nano particle for preparing also has naturally is conducive to carry out the experimental study relevant with animals and plants.Described amino acid with reproducibility refers to any in α-aminopropionic acid, histidine, tyrosine, tryptophan or the cysteine.
(2) measure the AgNO of 5-20 mL
3The aqueous solution (can measure according to actual needs to determine by volume, the at most volume that needs is got, suitability for industrialized production can enlarge in proportion), the polyvinylpyrrolidone (being designated hereinafter simply as PVP) that adds 0.05-0.2 g, the consumption of polyvinylpyrrolidone is determined according to the actual amount of reactant), guarantee that PVP concentration is at 5-20 mgmL
-1In the scope; The AgNO of general 0.001 mol
3Use the polyvinylpyrrolidone of 0.05-0.2 g to be advisable; After polyvinylpyrrolidone under agitation dissolves fully, add and AgNO
3The isopyknic amino acid solution with reproducibility of the aqueous solution stops stirring and allows reaction system be in static condition; PVP also is a kind of large molecule of biocompatibility, and the purpose that adopts PVP to stablize the Ag nano particle among the present invention is the biocompatibility that guarantees particle, does not have bio-toxicity.Described amino acid with reproducibility refers to any in α-aminopropionic acid, histidine, tyrosine, tryptophan or the cysteine.
(3) when the color of reaction system gradually becomes brown and optical clear (residing environment temperature was relevant when concrete required time carried out with reaction), nano particle generated and size less, centrifugal treating, the gained supernatant liquor is collected back the initial reaction container again, and the precipitation that obtains in the centrifuge tube bottom is stored in the deionized water stand-by.
(4) collected centrifugal supernatant liquor can continue reaction and again reach brown and optical clear state in put procedure, then adopts the method identical with step (3) again to realize separating of nano particle and reaction mother liquor; This step can repeatedly carry out repeatedly until centrifugal upper strata liquid no longer include the reaction carry out till (becoming brown).
(5) all previous separating obtained precipitation that is distributed in the deionized water is put together, and be settled to the AgNO that adopts when reacting initial
3The volume of the aqueous solution, can obtain concentration is 0.05-0.2 molL
-1The Ag nano-particle solution (this moment the Ag nano particle concentration and the AgNO that adopts
3The initial concentration of the aqueous solution is identical).
Described polyvinylpyrrolidone concentration is 5-20 mgmL
-1The concentration of PVP is too low, do not have the effect of dispersion and stabilized nanoscale particle, consumption is too many, the one, can not dissolve again after reaching capacity, the 2nd, the viscosity rise of whole system, the later stage centrifugal treating comparatively bothers, and adopts in addition too much PVP also can further not affect the final size of nano particle, causes on the contrary waste.
Described centrifugal treating is to carry out centrifugal about 10 minutes (such as 8-15 minute) under centrifuge 10000 rpm conditions.Centrifugation time can not be out centrifugal whole nano particles less than 8 minutes, was the waste to instrument and the energy above 15 minutes.In addition, centrifugal revolution is too low, can not realize the fully separation of nano particle, too highly also there is no need.
Obtain concentration by dilution and be respectively 100,200,300,400 or 500 μ molL
-1The Ag nano-particle solution.
Nano silver grain promotes the method for seed germination, it is characterized in that step is as follows:
(1) full, the uniform cucumber seeds of selection is clean with distilled water flushing, suction is for subsequent use; With the cucumber seeds grouping of rinsing well, process with the silver nano-particle solution of different quality concentration, under room temperature, soak 12 h; Wherein process in contrast with distilled water; Each processing arranges three repetitions.
(2) place culture dish to cultivate soaked cucumber seeds, the culture dish bottom is spread absorbent cotton and filter paper successively, and spills water-soaked filter paper, in 27 ± 1 ℃ of lower cultivations of insulating box; Every day observed and recorded Seed germination situation, and Taking Pictures recording.
When (3) cucumber seeds is cultivated 24 h, 48 h, 72 h, the germination number of statistics seed, the length of measurement bud; To calculate its germinating energy, germination percentage.
The result shows, Nano silver grain is processed the sprouting that can promote cucumber seeds.
Specifically, Nano silver grain promotes the method for seed germination, it is characterized in that:
(1) select 300 of full, uniform cucumber seeds, clean with distilled water flushing, blot for subsequent use with blotting paper; The cucumber seeds of rinsing well is divided into 6 groups, 50 every group, put into respectively 6 culture dishes and numbering, be respectively again the silver nano-particle solution processing of 0,100,200,300,400,500 μ mol/L with mass concentration, under room temperature, soak 12 h; Wherein process in contrast with distilled water; Each processing arranges three repetitions.
(2) place culture dish to cultivate soaked cucumber seeds, the culture dish bottom is spread absorbent cotton and filter paper successively, and spills water-soaked filter paper, in 27 ± 1 ℃ of lower cultivations of insulating box; Every day observed and recorded Seed germination situation, and Taking Pictures recording.
When (3) cucumber seeds is cultivated 24 h, 48 h, 72 h, the germination number of statistics seed, the length of measurement bud; To calculate its germinating energy, germination percentage.
The result shows, Nano silver grain is processed the sprouting that can promote cucumber seeds, and is that germinating energy and the germination percentage of cucumber seeds are best under the 300 μ mol/L treatment conditions in silver nano-particle solution concentration.
The detection method of metabolism variable effect in the procedure of Nano silver grain promotion seed germination is characterized in that step is as follows:
(1) Nano silver grain is processed the detection to water content in the Germination Process of Cucumber Seeds: respectively take out 20 seeds after the sprouting from each group, claim fresh weight W1, then it is placed on respectively and dries the W2 that weighs again in the air dry oven;
Or (2) Nano silver grain is processed the detection to protein content in the Germination Process of Cucumber Seeds: get germination seed 0.2 g, grind to form homogenate with 5 mL distilled water ice baths, move in the centrifuge tube, 4 ℃ of lower centrifugal 15 min of 12000 rpm, the gained supernatant is soluble protein liquid, get protein extract 1.0 mL, put into tool plug test tube, each sample repeats twice, add 5 mL G-250 solution, fully mix, place behind 2 min colorimetric under 595 nm, measure absorbance, check in protein content by calibration curve.
Or (3) Nano silver grain is processed the detection to peroxidase in the Germination Process of Cucumber Seeds (POD) enzymatic activity: get germination seed 0.5 g, with 10 mL lmol.L
-1PH8.0 Tris ice bath grinds to form homogenate, move in the centrifuge tube, 4 ℃ of lower centrifugal 15 min of 1500 rpm in the ultralow temperature centrifuge, the gained supernatant is zyme extract, get zyme extract 1.0 mL, put into tool plug test tube, each sample repeats twice, adds 2 mL benzidine acetic acid-sodium-acetate buffers, place again 28-32 ℃, insulation 3-5 min in the water-bath; During mensuration, add l mL 0.1mol. L
-1H
2O
2, shaking up immediately, and change in the cuvette, colorimetric under 470 nm is measured absorbance, from adding H
2O
2The time play timing, every 15s reading once,, calculate enzymatic activity.
Or (4) Nano silver grain is processed the detection to Total Soluble Sugar content in the Germination Process of Cucumber Seeds:
The extraction of Total Soluble Sugar: get germination seed 0.5 g, add 5-10 mL distilled water, fully grind, change in the scale test tube, plastic film sealing extracts 30 min, totally 2 times in boiling water, extracting liquid filtering enters 25 mL volumetric flasks, repeatedly washes test tube and residue, is settled to scale.
The mensuration of Total Soluble Sugar: get 0.5 mL sample liquid in test tube, repeat 2 times, adding distil water 1.5 mL add 1 mL, 9% phenol solution in test tube, shake up, and add the 5 mL concentrated sulfuric acids, and the color solution cumulative volume is 8 mL, place 30 min under constant temperature; Colour developing is in 485 nm wavelength colorimetric estimations.
Or (5) Nano silver grain is processed the detection to total amylase and alpha-amylase activity in the Germination Process of Cucumber Seeds: adopt 3,5-dinitrosalicylic Acid Colorimetry (DNS).Peroxidase in the same step of extracting method (3); Accurately drawing step (3) extracts in 10 mL to two clean test tubes of enzyme liquid, in 40 ℃ of water-baths, extract l h, constantly stir therebetween, after insulation finishes, filter in 25 mL test tubes, and with 40 ℃ of warm water drip washing filter residues, be settled to scale after the cooling, insulation 15 min in 70 ℃ of waters bath with thermostatic control with passivation p-amylase, obtain the AMS crude extract; Enzyme liquid is for subsequent use under being stored in 4 ℃.
Accurately draw 2 mL enzyme liquid to clean tube, put into immediately boiling water bath with one and boil 15 min, cool to room temperature is processed in contrast; Then add respectively l ml 1% soluble starch solution to other test tubes, shake up, insulation 20 min in 37 ℃ of waters bath with thermostatic control; Insulation changes two test tubes over to immediately and boils 15 min in the boiling water bath after finishing, and stops reaction; From each test tube, take out respectively l mL amylorrhexis liquid, move in the empty test tube of corresponding numbering, add 2 mL DNS reagent, reaction 5 min in the boiling water bath, cooling, adding distil water 7 mL, through the contrast that is treated to of water-bath 15 min, measure light absorption value with first at 520 nm places.
Compared with the prior art the present invention has following advantage:
1. adopted alanine to prepare Nano silver grain as reducing agent.Because alanine is a seed amino acid, itself is exactly a kind of biomolecule, therefore has good biocompatibility.In addition in preparation process the rate of reduction of alanine to compare to the strong reductants such as sodium borohydride commonly used more gentle, easily realize control by stages.
2. adopted PVP to prevent the gathering of nano particle as dispersant.PVP itself also is the good high polymer of a kind of biocompatibility, often is applied in the middle of skin care item or even the food.
3. whole preparation is less demanding to reaction condition, does not also need special instrument and equipment, therefore is easier to accomplish scale production.
4. the investigation Nano silver grain still belongs to the first time on the research work of the impact of seed germination and plant growth, and the research Nano silver grain is had certain directive significance in the botany application facet.
5. experimental result shows seed more easily sprouting with respect to control group of processing with Nano silver grain, plant is more sturdy, root system is more flourishing, what is particularly worth mentioning is that pest-resistant harmful ability strengthens, and illustrates that nano particle provided by the present invention is expected to be applied in the agricultural production.
Below in conjunction with embodiment embodiments of the present invention are described in further detail.
The specific embodiment
Embodiment 1
(1) under 15 ℃ of conditions of environment temperature, measures 20 mL, 0.1 molL
-1AgNO
3The aqueous solution adds 0.2 g PVP powder and continues to be stirred to fully dissolving under stirring.Measure 20 mL, 0.1 molL
-1The α-aminopropionic acid aqueous solution under agitation add above solution, then stop to stir, leave standstill reaction system.
(2) leave standstill 10 minutes after, reaction system changes dark brown optical clear solution gradually into, illustrating has had undersized nano particle to form this moment.At once with this solution with supercentrifuge centrifugation 10 minutes under the rotating speed of 1000rpm/min, lurid upper strata liquid is refunded reaction vessel, can get the Nano silver grain of size about 20 nanometers in the centrifuge tube bottom.
(3) for the first time the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 30 minutes, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(4) for the second time the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 1 hour, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(5) the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 12 hours for the third time, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
The upper strata liquid of (6) the 4th time centrifugation gained leaves standstill about 72 hours and makes it complete reaction, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(7) all previous separating obtained precipitation is distributed to can to obtain concentration in the deionized water of 20 mL be 0.1 molL
-1The Ag nano-particle solution, can obtain the Ag nano-particle solution that concentration be respectively 100,200,300,400,500 μ mol/L by dilution on this basis.
(8) select 300 of full, uniform cucumber seeds, clean with distilled water flushing, blot for subsequent use with blotting paper.The cucumber seeds of rinsing well is divided into 6 groups, 50 every group, put into respectively 6 culture dishes and numbering, be respectively again the silver nano-particle solution processing of 0,100,200,300,400,500 μ mol/L with concentration, under room temperature, soak 12 h.Wherein process in contrast with distilled water.Each processing arranges three repetitions.
(9) place culture dish to cultivate soaked cucumber seeds, the culture dish bottom is spread absorbent cotton and filter paper successively, and spills an amount of water (suitable to soak filter paper), in 27 ± 1 ℃ of lower cultivations of insulating box.Check between culture period whether the moisture content in the culture dish is sufficient, gives certain replenishing.Every day observed and recorded Seed germination situation, and Taking Pictures recording.
When (10) cucumber seeds is cultivated 24 h, 48 h, 72 h, the germination number of statistics seed (being as the criterion with prominent breaking in the seed coat), the length of measurement bud.Calculating its germinating energy, germination percentage, the result shows, Nano silver grain is processed the sprouting that can promote cucumber seeds, and under 300 μ mol/L treatment conditions, germinating energy and the germination percentage of cucumber seeds best (better).
(11) get respectively cucumber seeds after the sprouting of processing with the Nano silver grain aqueous solution of clear water and optium concentration (300 μ mol/L), be seeded in the nutritive cube, each nutritive cube is broadcast 3 seeds, routine observation growth of seedling situation, when treating that cucumber grows 4 true leaves, adopt the plant height of tape measure cucumber, thick with the vernier caliper measurement Cucumis sativus stem; Measure the cucumber leaf area with the template weight method, adopt oven drying method to measure the dry matter content of cucumber leaves relative water content and plant.
Any replacement in this example in α-aminopropionic acid available set propylhomoserin, tyrosine, tryptophan or the cysteine.
Embodiment 2
(1) under 15 ℃ of conditions of environment temperature, measures 20 mL, 0.1 molL
-1AgNO
3The aqueous solution adds 0.1 g PVP powder and continues to be stirred to fully dissolving under stirring.Measure 20 mL, 0.1 molL
-1The α-aminopropionic acid aqueous solution under agitation add above solution, then stop to stir, leave standstill reaction system.
(2) leave standstill 10 minutes after, reaction system changes dark brown optical clear solution gradually into, illustrating has had undersized nano particle to form this moment.At once with this solution with supercentrifuge centrifugation 10 minutes under the rotating speed of 1000rpm/min, lurid upper strata liquid is refunded reaction vessel, can get the Nano silver grain of size about 20 nanometers in the centrifuge tube bottom.
(3) for the first time the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 30 minutes, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(4) for the second time the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 1 hour, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(5) the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 12 hours for the third time, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
The upper strata liquid of (6) the 4th time centrifugation gained leaves standstill about 72 hours and makes it complete reaction, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(7) all previous separating obtained precipitation is distributed to can to obtain concentration in the deionized water of 20 mL be 0.1 molL
-1The Ag nano-particle solution, can obtain the Ag nano-particle solution that concentration be respectively 100,200,300,400,500 μ mol/L by dilution on this basis.
(8) select 300 of full, uniform cucumber seeds, clean with distilled water flushing, blot for subsequent use with blotting paper.The cucumber seeds of rinsing well is divided into 6 groups, 50 every group, put into respectively 6 culture dishes and numbering, be respectively again the silver nano-particle solution processing of 0,100,200,300,400,500 μ mol/L with concentration, under room temperature, soak 12 h.Wherein process in contrast with distilled water.Each processing arranges three repetitions.Any replacement in above-mentioned α-aminopropionic acid available set propylhomoserin, tyrosine, tryptophan or the cysteine.
(9) place culture dish to cultivate soaked cucumber seeds, the culture dish bottom is spread absorbent cotton and filter paper successively, and spills an amount of water (suitable to soak filter paper), in 27 ± 1 ℃ of lower cultivations of insulating box.Check between culture period whether the moisture content in the culture dish is sufficient, gives certain replenishing.Every day observed and recorded Seed germination situation, and Taking Pictures recording.
When (10) cucumber seeds is cultivated 24 h, 48 h, 72 h, the germination number of statistics seed (being as the criterion with prominent breaking in the seed coat), the length of measurement bud.Calculating its germinating energy, germination percentage, the result shows, Nano silver grain is processed the sprouting that can promote cucumber seeds, and under 300 μ mol/L treatment conditions, germinating energy and the germination percentage of cucumber seeds are best.
(11) from each group, respectively take out 20 seeds after the sprouting, claim fresh weight W1, then it is placed on respectively and dries the W2 that weighs again in the air dry oven, then calculate the Nano silver grain processing to the impact of water content in the Germination Process of Cucumber Seeds.
(12) get germination seed 0.2 g, grind to form homogenate with 5 mL distilled water ice baths, move in the centrifuge tube, 4 ℃ of lower centrifugal 15 min of 12000 rpm, the gained supernatant is soluble protein liquid, get protein extract 1.0 mL, put into tool plug test tube (each sample repeats twice), add 5 mL G-250 solution, fully mix, place behind 2 min colorimetric under 595 nm, measure absorbance, check in protein content by calibration curve, obtain the Nano silver grain processing to the data that affect of protein content in the Germination Process of Cucumber Seeds.
(13) get germination seed 0.5 g, with 10 mL lmol.L
-1, pH8.0 Tris ice bath grinds to form homogenate, moves in the centrifuge tube, and 4 ℃ of lower centrifugal 15 min of 1500 rpm in the ultralow temperature centrifuge, the gained supernatant is zyme extract.Get zyme extract 1.0 mL, put into tool plug test tube (each sample repeats twice), add 2 mL benzidine acetic acid, one sodium-acetate buffer, place again 28-32 ℃, insulation 3-5 min in the water-bath.During mensuration, add l mL 0.1mol. L
-1H
2O
2, shaking up immediately, and change in the cuvette, colorimetric under 470 nm is measured absorbance (from adding H
2O
2The time play timing, every 15s reading is once), calculate enzymatic activity.Obtain the Nano silver grain processing to the data that affect of peroxidase in the Germination Process of Cucumber Seeds (POD) enzymatic activity.
(14) get germination seed 0.5 g, add 5-10 mL distilled water, fully grind, change in the scale test tube, plastic film sealing extracts 30 min in boiling water, and totally 2 times, extracting liquid filtering enters 25 mL volumetric flasks, repeatedly washes test tube and residue, is settled to scale.
Get the above-mentioned sample liquid of 0.5 mL and (repeat 2 times) in test tube, adding distil water 1.5 mL add 1 mL, 9% phenol solution in test tube, shake up, and add the 5 mL concentrated sulfuric acids, and the color solution cumulative volume is 8 mL, place 30 min under constant temperature.Colour developing is in 485 nm wavelength colorimetric estimations.Thereby measure the Nano silver grain processing to the impact of Total Soluble Sugar content in the Germination Process of Cucumber Seeds.
(15) adopt 3,5-dinitrosalicylic Acid Colorimetry (DNS).The same peroxidase of extracting method.Accurately drawing step (3) extracts in 10 mL to two clean test tubes of enzyme liquid, in 40 ℃ of water-baths, extract l h,, constantly stir therebetween, after insulation finishes, filter in 25 mL test tubes, and with 40 ℃ of warm water drip washing filter residues, be settled to scale after the cooling, insulation 15 min in 70 ℃ of waters bath with thermostatic control, with passivation p-amylase, obtain the AMS crude extract.Enzyme liquid is for subsequent use under being stored in 4 ℃.
Accurately draw 2 mL enzyme liquid to clean tube, put into immediately boiling water bath with one and boil 15 min, cool to room temperature is processed in contrast.Then add respectively l ml 1% soluble starch solution to other test tubes, shake up, insulation 20 min in 37 ℃ of waters bath with thermostatic control; Insulation changes two test tubes over to immediately and boils 15 min in the boiling water bath after finishing, and stops reaction.From each test tube, take out respectively l mL amylorrhexis liquid, move in the empty test tube of corresponding numbering, add 2 mL DNS reagent, reaction 5 min in the boiling water bath, cooling, adding distil water 7 mL, through the contrast that is treated to of water-bath 15 min, measure light absorption value with first at 520 nm places.Obtain the Nano silver grain processing to the data that affect of total amylase and alpha-amylase activity in the Germination Process of Cucumber Seeds.
Embodiment 3
(1) under 25 ℃ of conditions of environment temperature, measures 20 mL, 0.1 molL
-1AgNO
3The aqueous solution adds 0.2 g PVP powder and continues to be stirred to fully dissolving under stirring.Measure 20 mL, 0.1 molL
-1The α-aminopropionic acid aqueous solution under agitation add above solution, then stop to stir, leave standstill reaction system.
(2) leave standstill 5 minutes after, reaction system changes dark brown optical clear solution gradually into, illustrating has had undersized nano particle to form this moment.At once with this solution with supercentrifuge centrifugation 10 minutes under the rotating speed of 1000rpm/min, lurid upper strata liquid is refunded reaction vessel, can get the Nano silver grain of size about 20 nanometers in the centrifuge tube bottom.
(3) for the first time the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 15 minutes, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(4) for the second time the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 0.5 hour, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(5) the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 6 hours for the third time, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
The upper strata liquid of (6) the 4th time centrifugation gained leaves standstill about 36 hours and makes it complete reaction, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(7) all previous separating obtained precipitation is distributed to can to obtain concentration in the deionized water of 20 mL be 0.1 molL
-1The Ag nano-particle solution.Can obtain the Ag nano-particle solution that concentration is respectively 100,200,300,400,500 μ mol/L by dilution on this basis.
Step (7)-(10) are with embodiment 1(7)-(10).Any replacement in above-mentioned α-aminopropionic acid available set propylhomoserin, tyrosine, tryptophan or the cysteine.
Embodiment 4
(1) under 25 ℃ of conditions of environment temperature, measures 20 mL, 0.1 molL
-1AgNO
3The aqueous solution adds 0.1 g PVP powder and continues to be stirred to fully dissolving under stirring.Measure 20 mL, 0.1 molL
-1The α-aminopropionic acid aqueous solution under agitation add above solution, then stop to stir, leave standstill reaction system.
(2) leave standstill 5 minutes after, reaction system changes dark brown optical clear solution gradually into, illustrating has had undersized nano particle to form this moment.At once with this solution with supercentrifuge centrifugation 10 minutes under the rotating speed of 1000rpm/min, lurid upper strata liquid is refunded reaction vessel, can get the Nano silver grain of size about 20 nanometers in the centrifuge tube bottom.
(3) for the first time the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 15 minutes, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(4) for the second time the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 0.5 hour, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(5) the upper strata liquid of centrifugation gained leaves standstill and again is converted into dark brown optical clear solution about 6 hours for the third time, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
The upper strata liquid of (6) the 4th time centrifugation gained leaves standstill about 36 hours and makes it complete reaction, adopts the step identical with step (2) again to separate and again obtains the Nano silver grain of size about 20 nanometers.
(7) all previous separating obtained precipitation is distributed to can to obtain concentration in the deionized water of 20 mL be 0.1 molL
-1The Ag nano-particle solution.Can obtain the Ag nano-particle solution that concentration is respectively 100,200,300,400,500 μ mol/L by dilution on this basis.Any replacement in above-mentioned α-aminopropionic acid available set propylhomoserin, tyrosine, tryptophan or the cysteine.
Step (7)-(14) are with embodiment 1(7)-(14).
Embodiment 5
(1) under 15 ℃ of conditions of environment temperature, measures 20 mL, 0.2 molL
-1AgNO
3The aqueous solution adds 0.4 g PVP powder and continues to be stirred to fully dissolving under stirring.Measure 20 mL, 0.2 molL
-1The α-aminopropionic acid aqueous solution under agitation add above solution, then stop to stir, leave standstill reaction system.
(2) leave standstill 10 minutes after, reaction system changes dark brown optical clear solution gradually into, illustrating has had undersized nano particle to form this moment.At once with this solution with supercentrifuge centrifugation 10 minutes under the rotating speed of 1000rpm/min, lurid upper strata liquid is refunded reaction vessel, can get the Nano silver grain of size about 20 nanometers in the centrifuge tube bottom.
Step (3)-(5) are identical with step (3)-(5) among the embodiment 1.
(6) all previous separating obtained precipitation is distributed to can to obtain concentration in the deionized water of 20 mL be 0.1 molL
-1The Ag nano-particle solution.Can obtain the Ag nano-particle solution that concentration is respectively 100,200,300,400,500 μ mol/L by dilution on this basis.
Step (7)-(10) are with embodiment 1(7)-(10).Any replacement in above-mentioned α-aminopropionic acid available set propylhomoserin, tyrosine, tryptophan or the cysteine.
Embodiment 6
(1) under 15 ℃ of conditions of environment temperature, measures 20 mL, 0.1 molL
-1AgNO
3The aqueous solution adds 0.4 g PVP powder and continues to be stirred to fully dissolving under stirring.Measure 20 mL, 0.1 molL
-1The α-aminopropionic acid aqueous solution under agitation add above solution, then stop to stir, leave standstill reaction system.
(2) leave standstill 10 minutes after, reaction system changes dark brown optical clear solution gradually into, illustrating has had undersized nano particle to form this moment.At once with this solution with supercentrifuge centrifugation 10 minutes under the rotating speed of 1000rpm/min, lurid upper strata liquid is refunded reaction vessel, can get the Nano silver grain of size about 20 nanometers in the centrifuge tube bottom.
Step (3)-(5) are identical with step (3)-(5) among the embodiment 1.
(6) all previous separating obtained precipitation is distributed to can to obtain concentration in the deionized water of 20 mL be 0.1 molL
-1The Ag nano-particle solution.Can obtain the Ag nano-particle solution that concentration is respectively 100,200,300,400,500 μ mol/L by dilution on this basis.
Step (7)-(14) are with embodiment 1(7)-(14).Any replacement in above-mentioned α-aminopropionic acid available set propylhomoserin, tyrosine, tryptophan or the cysteine.
Claims (9)
1. the preparation method of a Nano silver grain is characterized in that step is as follows:
Get AgNO
3The aqueous solution adds polyvinylpyrrolidone, dissolves rear adding and AgNO fully until polyvinylpyrrolidone
3The isopyknic amino acid solution with reproducibility of the aqueous solution, when the color of reaction system gradually becomes brown and optical clear, nano particle generated and size less, centrifugal treating, the gained supernatant liquor is collected back the initial reaction container again, the precipitation that obtains in the centrifuge tube bottom is stored in deionized water, obtains the Ag nano-particle solution.
2. according to the preparation method of nano particle claimed in claim 1, it is characterized in that step is as follows:
(1) under temperature 15-25 ℃ of condition, configuration concentration is the AgNO of 0.05-0.2 molL-1 respectively
3The aqueous solution and concentration 0.05-0.2 molL
-1Amino acid with reproducibility;
(2) measure the AgNO of 5-20 mL
3The aqueous solution, the polyvinylpyrrolidone of adding 0.05-0.2 g guarantees that PVP concentration is at 5-20 mgmL
-1In the scope; After polyvinylpyrrolidone under agitation dissolves fully, add and AgNO
3The isopyknic amino acid solution with reproducibility of the aqueous solution stops stirring and allows reaction system be in static condition; Described amino acid with reproducibility refers to any in α-aminopropionic acid, histidine, tyrosine, tryptophan or the cysteine;
(3) when the color of reaction system gradually becomes brown and optical clear, nano particle generated and size less, centrifugal treating, the gained supernatant liquor is collected back the initial reaction container again, and the precipitation that obtains in the centrifuge tube bottom is stored in the deionized water stand-by;
(4) collected centrifugal supernatant liquor can continue reaction and again reach brown and optical clear state in put procedure, then adopts the method identical with step (3) again to realize separating of nano particle and reaction mother liquor; This step can repeatedly carry out repeatedly until centrifugal upper strata liquid no longer include the reaction carry out till;
(5) all previous separating obtained precipitation that is distributed in the deionized water is put together, and be settled to the AgNO that adopts when reacting initial
3The volume of the aqueous solution, can obtain concentration is 0.05-0.2 molL
-1The Ag nano-particle solution.
3. by the preparation method of nano particle claimed in claim 2, it is characterized in that centrifugal treating be under centrifuge 10000 rpm conditions, carry out 10 minutes centrifugal.
4. by the preparation method of nano particle claimed in claim 2, it is characterized in that obtaining concentration by dilution is respectively 100,200,300,400 or 500 μ molL
-1The Ag nano-particle solution.
5. method of utilizing claim 2 or 4 described Nano silver grains to promote seed germinations is characterized in that step is as follows:
(1) full, the uniform cucumber seeds of selection is clean with distilled water flushing, suction is for subsequent use; With the cucumber seeds grouping of rinsing well, process with the silver nano-particle solution of different quality concentration, under room temperature, soak 12 h; Wherein process in contrast with distilled water; Each processing arranges three repetitions;
(2) place culture dish to cultivate soaked cucumber seeds, the culture dish bottom is spread absorbent cotton and filter paper successively, and spills water-soaked filter paper, in 27 ± 1 ℃ of lower cultivations of insulating box; Every day observed and recorded Seed germination situation, and Taking Pictures recording;
When (3) cucumber seeds is cultivated 24 h, 48 h, 72 h, the germination number of statistics seed, the length of measurement bud; To calculate its germinating energy, germination percentage;
The result shows, Nano silver grain is processed the sprouting that can promote cucumber seeds.
6. promote the method for seed germination according to Nano silver grain claimed in claim 5, it is characterized in that:
(1) select 300 of full, uniform cucumber seeds, clean with distilled water flushing, blot for subsequent use with blotting paper; The cucumber seeds of rinsing well is divided into 6 groups, 50 every group, put into respectively 6 culture dishes and numbering, be respectively 0,100,200,300,400,500 μ molL with mass concentration again
-1Silver nano-particle solution process, under room temperature, soak 12 h; Wherein process in contrast with distilled water; Each processing arranges three repetitions;
(2) place culture dish to cultivate soaked cucumber seeds, the culture dish bottom is spread absorbent cotton and filter paper successively, and spills water-soaked filter paper, in 27 ± 1 ℃ of lower cultivations of insulating box; Every day observed and recorded Seed germination situation, and Taking Pictures recording;
When (3) cucumber seeds is cultivated 24 h, 48 h, 72 h, the germination number of statistics seed, the length of measurement bud; To calculate its germinating energy, germination percentage;
The result shows, Nano silver grain is processed the sprouting that can promote cucumber seeds, and is that germinating energy and the germination percentage of cucumber seeds are best under the 300 μ mol/L treatment conditions in silver nano-particle solution concentration.
7. detection method of utilizing metabolism variable effect in the procedure that Nano silver grain claimed in claim 6 promotes seed germination is characterized in that step is as follows:
(1) Nano silver grain is processed the detection to water content in the Germination Process of Cucumber Seeds: respectively take out 20 seeds after the sprouting from each group, claim fresh weight W1, then it is placed on respectively and dries the W2 that weighs again in the air dry oven;
Or (2) Nano silver grain is processed the detection to protein content in the Germination Process of Cucumber Seeds: get germination seed 0.2 g, grind to form homogenate with 5 mL distilled water ice baths, move in the centrifuge tube, 4 ℃ of lower centrifugal 15 min of 12000 rpm, the gained supernatant is soluble protein liquid, get protein extract 1.0 mL, put into tool plug test tube, each sample repeats twice, add 5 mL G-250 solution, fully mix, place behind 2 min colorimetric under 595 nm, measure absorbance, check in protein content by calibration curve;
Or (3) Nano silver grain is processed the detection to peroxidase enzymatic activity in the Germination Process of Cucumber Seeds: get germination seed 0.5 g, with 10 mL lmol.L
-1PH8.0 Tris ice bath grinds to form homogenate, move in the centrifuge tube, 4 ℃ of lower centrifugal 15 min of 1500 rpm in the ultralow temperature centrifuge, the gained supernatant is zyme extract, get zyme extract 1.0 mL, put into tool plug test tube, each sample repeats twice, adds 2 mL benzidine acetic acid-sodium-acetate buffers, place again 28-32 ℃, insulation 3-5 min in the water-bath; During mensuration, add l mL 0.1mol. L
-1H
2O
2, shaking up immediately, and change in the cuvette, colorimetric under 470 nm is measured absorbance, from adding H
2O
2The time play timing, every 15s reading once,, calculate enzymatic activity;
Or (4) Nano silver grain is processed the detection to Total Soluble Sugar content in the Germination Process of Cucumber Seeds:
The extraction of Total Soluble Sugar: get germination seed 0.5 g, add 5-10 mL distilled water, fully grind, change in the scale test tube, plastic film sealing extracts 30 min, totally 2 times in boiling water, extracting liquid filtering enters 25 mL volumetric flasks, repeatedly washes test tube and residue, is settled to scale;
The mensuration of Total Soluble Sugar: get 0.5 mL sample liquid in test tube, repeat 2 times, adding distil water 1.5 mL add 1 mL, 9% phenol solution in test tube, shake up, and add the 5 mL concentrated sulfuric acids, and the color solution cumulative volume is 8 mL, place 30 min under constant temperature; Colour developing is in 485 nm wavelength colorimetric estimations;
Or (5) Nano silver grain is processed the detection to total amylase and alpha-amylase activity in the Germination Process of Cucumber Seeds: adopt 3,5-dinitrosalicylic Acid Colorimetry; Peroxidase in the same step of extracting method (3); Accurately drawing step (3) extracts in 10 mL to two clean test tubes of enzyme liquid, in 40 ℃ of water-baths, extract l h, constantly stir therebetween, after insulation finishes, filter in 25 mL test tubes, and with 40 ℃ of warm water drip washing filter residues, be settled to scale after the cooling, insulation 15 min in 70 ℃ of waters bath with thermostatic control with passivation p-amylase, obtain the AMS crude extract; Enzyme liquid is for subsequent use under being stored in 4 ℃;
Accurately draw 2 mL enzyme liquid to clean tube, put into immediately boiling water bath with one and boil 15 min, cool to room temperature is processed in contrast; Then add respectively l ml 1% soluble starch solution to other test tubes, shake up, insulation 20 min in 37 ℃ of waters bath with thermostatic control; Insulation changes two test tubes over to immediately and boils 15 min in the boiling water bath after finishing, and stops reaction; From each test tube, take out respectively l mL amylorrhexis liquid, move in the empty test tube of corresponding numbering, add 2 mL DNS reagent, reaction 5 min in the boiling water bath, cooling, adding distil water 7 mL, through the contrast that is treated to of water-bath 15 min, measure light absorption value with first at 520 nm places.
8. according to the preparation method of nano particle claimed in claim 2, it is characterized in that step is as follows:
(1) under 15 ℃ of conditions of environment temperature, measures 20 mL, 0.1 molL
-1AgNO
3The aqueous solution adds 0.2 g PVP powder and continues to be stirred to fully dissolving under stirring; Measure 20 mL, 0.1 molL
-1The α-aminopropionic acid aqueous solution under agitation add above solution, then stop to stir, leave standstill reaction system;
(2) leave standstill 10 minutes after, reaction system changes dark brown optical clear solution gradually into, at once with this solution with supercentrifuge centrifugation 10 minutes under the rotating speed of 1000rpm/min, lurid upper strata liquid is refunded reaction vessel, can get size at the Nano silver grain of 20 nanometers in the centrifuge tube bottom;
(3) for the first time the upper strata liquid of centrifugation gained leaves standstill and again was converted into dark brown optical clear solution in 30 minutes, adopts the step identical with step (2) again to separate and again obtains size at the Nano silver grain of 20 nanometers;
(4) for the second time the upper strata liquid of centrifugation gained leaves standstill and again was converted into dark brown optical clear solution in 1 hour, adopts the step identical with step (2) again to separate and again obtains size at the Nano silver grain of 20 nanometers;
(5) the upper strata liquid of centrifugation gained leaves standstill and again was converted into dark brown optical clear solution in 12 hours for the third time, adopts the step identical with step (2) again to separate and again obtains size at the Nano silver grain of 20 nanometers;
The upper strata liquid of (6) the 4th centrifugation gained leaves standstill and made it complete reaction in 72 hours, adopts the step identical with step (2) again to separate and again obtains size at the Nano silver grain of 20 nanometers;
(7) all previous separating obtained precipitation is distributed to can to obtain concentration in the deionized water of 20 mL be 0.1 molL
-1The Ag nano-particle solution, can obtain concentration by dilution on this basis and be respectively 100,200,300,400,500 μ molL
-1The Ag nano-particle solution.
9. promote the method for seed germination according to Nano silver grain claimed in claim 5, it is characterized in that step is as follows:
(1) select 300 of full, uniform cucumber seeds, clean with distilled water flushing, blot for subsequent use with blotting paper; The cucumber seeds of rinsing well is divided into 6 groups, 50 every group, put into respectively 6 culture dishes and numbering, be respectively 0,100,200,300,400,500 μ molL with concentration again
-1Silver nano-particle solution process, under room temperature, soak 12 h; Wherein process in contrast with distilled water; Each processing arranges three repetitions;
(2) place culture dish to cultivate soaked cucumber seeds, the culture dish bottom is spread absorbent cotton and filter paper successively, and it is suitable to soak filter paper to spill water, in 27 ± 1 ℃ of lower cultivations of insulating box; Every day observed and recorded Seed germination situation, and Taking Pictures recording;
When (3) cucumber seeds is cultivated 24 h, 48 h, 72 h, the germination number of statistics seed, the length of measurement bud; Calculating its germinating energy, germination percentage, the result shows, Nano silver grain is processed the sprouting that can promote cucumber seeds, and under 300 μ mol/L treatment conditions, germinating energy and the germination percentage of cucumber seeds are better.
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| CN113940361A (en) * | 2021-11-18 | 2022-01-18 | 陕西理工大学 | Core-shell type magnetic nano Fe3O4Preparation method of/Cu/CuO @ Ag composite material |
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