CN109879329A - A kind of preparation method of the nanometer magnetic bead for ultramicron nucleic acid extraction - Google Patents
A kind of preparation method of the nanometer magnetic bead for ultramicron nucleic acid extraction Download PDFInfo
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- CN109879329A CN109879329A CN201910239179.9A CN201910239179A CN109879329A CN 109879329 A CN109879329 A CN 109879329A CN 201910239179 A CN201910239179 A CN 201910239179A CN 109879329 A CN109879329 A CN 109879329A
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Abstract
The present invention relates to a kind of preparation methods of nano biological magnetic bead for ultramicron nucleic acid extraction, belong to field of biotechnology, the following steps are included: first, prepare nano ferriferrous oxide dispersion liquid: take nano ferriferrous oxide powder successively through ethyl alcohol and containing surfactant aqueous solution washing after, it is dispersed in the ethanol solution containing oleic acid, it is handled again with homogenizer, obtains nano ferriferrous oxide dispersion liquid；Secondly, silica is wrapped up on nano ferriferrous oxide surface: nano ferriferrous oxide dispersion liquid is put into reaction kettle, tetraethyl orthosilicate and ammonia water mixture reaction is added, successively the aqueous solution with alcohol solution and containing surfactant washs, it is dispersed in the aqueous solution containing surfactant, obtains magnetic bead solution；Magnetic bead solution, ultrasonic agitation are handled with homogenizer, water washing to neutrality is stored in ultrapure water, obtains nano biological magnetic bead.Its simple process, magnetic responsiveness can be strong, and the adsorbed hydroxyl content is abundant, can be used for the extraction of ultramicron sample of nucleic acid, and DNA yield is high.
Description
Technical field
The invention belongs to field of biotechnology, and in particular, to a kind of nano biological magnetic for ultramicron nucleic acid extraction
The preparation method of pearl.
Background technique
Constantly improve and develop with DNA extractive technique, paramagnetic particle method extractive technique is slowly risen, the core as extraction
Material magnetic bionanoparticles are gradually known.Because DNA extraction is the first step of all follow-up studies, DNA is mentioned
The efficiency and effect taken directly affects subsequent research.Magnetic bionanoparticles are to pass through chemistry by magnetic material and high molecular material
Or a kind of functional material with numerous excellent properties that physical method is combined.
A kind of novel magnetic materials that magnetic bionanoparticles get up as developed recently not only have nano effect, i.e. table
Face effect, small-size effect, macro quanta tunnel effect and bulk effect also have magnetic property, such as superparamagnetism and high magnetic
The characteristics such as rate.Its application develops to high-technology field from traditional technical field, extends from simple magnetics range
To interdisciplinary field relevant to magnetics.It is most widely used with nucleic acid separation field.
Although magnetic bionanoparticles using increasingly extensive, but still have certain problems in biomedicine field: external
The magnetic bionanoparticles of brand are in biological field using more mature, but its price is higher, is valuated with milliliter, usually domestic
5 ~ 6 times, core technology of preparing of the domestic magnetic bead due to that cannot grasp production magnetic bead, the nanometer magnetic bead particle size of preparation has centainly
Size distribution, and poor biocompatibility, moreover, being difficult to keep there is also ferromagnetism in bad dispersibility, modifying process and function
The disadvantages of change degree is relatively low.
Summary of the invention
In order to solve deficiency in the prior art, the purpose of the present invention is to provide a kind of for ultramicron nucleic acid extraction
The preparation method of nano biological magnetic bead, process flow is simple, easy to operate, is suitable for industrial mass production；It is obtained
Nano biological magnetic bead is core-shell structure, and the adsorbed hydroxyl content is abundant, dispersed excellent, every mg magnetic bead strong with DNA binding ability
In combination with the DNA for being not less than 10ug, it can be used in the extraction of ultramicron sample of nucleic acid, improve the detection to ultramicron sample of nucleic acid
Sensitivity.
To achieve the goals above, the present invention use the specific scheme is that
A kind of preparation method of the nano biological magnetic bead for ultramicron nucleic acid extraction, it includes following steps:
Step 1: nano ferriferrous oxide dispersion liquid preparation
100g~10000g nano ferriferrous oxide powder is taken to be washed again with the aqueous solution containing surfactant after ethanol washing
It washs, is then dispersed in 5~10L and contains in the ethanol water of oleic acid, obtain nano ferriferrous oxide primary solution；By four oxygen of nanometer
Change three-iron primary solution to be handled with high pressure homogenizer, the high pressure homogenizer running parameter is 80MPa~150MPa, number is handled
It is 1~6 time, obtains nano ferriferrous oxide dispersion liquid；
Step 2: silica is wrapped up on nano ferriferrous oxide surface
Nano ferriferrous oxide dispersion liquid obtained by step 1 is put into reaction kettle, being added includes tetraethyl orthosilicate and ammonium hydroxide
100~2000ml of mixed liquor reacts 1~5h, is washed again with the aqueous solution containing surfactant after being washed with alcohol solution, most
It is dispersed in the aqueous solution containing surfactant afterwards, obtains magnetic bead solution；By gained magnetic bead solution with high pressure homogenizer handle 1~
2 times, the high pressure homogenizer running parameter is 60~120MPa, and after treatment continues ultrasonic agitation 12~for 24 hours, ultrasonic agitation
In the process control temperature be no more than 30 DEG C, be then in neutrality with milli-Q water to solution PH, be stored in ultrapure water to get with
In the nano biological magnetic bead of ultramicron nucleic acid extraction.
Advanced optimized as to above scheme, the nano ferriferrous oxide powder be by Hydrolyze method, hydro-thermal method,
Made from coprecipitation or ball-milling method.
It is advanced optimized as to above scheme, surface is living in the aqueous solution described in step 1 containing surfactant
Property agent volume fraction be 1%；The surfactant logical, nonylphenol polyoxyethylene ether, vegetable oil, citric acid, 12 for Qula
The mixture of one or more of sodium alkyl sulfonate and cetyl trimethylammonium bromide.Further, the vegetable oil
For maize germ oil.
It is advanced optimized as to above scheme, surface is living in the aqueous solution described in step 2 containing surfactant
Property agent volume fraction be 1%-5%；The surfactant logical, polyethylene glycol, glycerol, citric acid, dodecyl sulphur for Qula
The mixture of one or more of sour sodium and cetyl trimethylammonium bromide.
It is advanced optimized as to above scheme, the partial size of nano ferriferrous oxide powder described in step 1 is 10nm-
500nm, saturation magnetization are not less than 50 emu/g.
Advanced optimized as to above scheme, in the ethanol water containing oleic acid the content of oleic acid be 1~
100ml。
It is advanced optimized as to above scheme, ultrasonic power described in step 2 is not less than 800W.
It is advanced optimized as to above scheme, includes the mixed liquor of tetraethyl orthosilicate and ammonium hydroxide described in step 2
The ratio of middle tetraethyl orthosilicate and ammonium hydroxide is 0.2~1.0.
It is advanced optimized as to above scheme, alcohol is ethyl alcohol, isopropanol, the third three in alcohol solution described in step 2
The compound of one or more of alcohol；The concentration of alcohol is not less than 50% in the alcohol solution.
The utility model has the advantages that
1, the process flow of preparation method of the present invention is simple, easy to operate, is suitable for industrial mass production；It is obtained
Nano biological magnetic bead is core-shell structure, and magnetic responsiveness can be strong, and the adsorbed hydroxyl content is abundant, dispersed excellent, the energy in conjunction with DNA
Power is strong, can be used in the extraction of ultramicron sample of nucleic acid, and DNA yield is high, and can improve the spirit of the detection to ultramicron sample of nucleic acid
Sensitivity.
2, the adsorbed hydroxyl content for the nano biological magnetic bead being prepared using preparation method of the present invention is not less than
0.2mmol/g can not only be effectively improved its dispersion stabilization, and since surface is rich in active hydroxyl convenient for by anti-
It should be in conjunction with several functional molecules；Uniform particle sizes, average grain diameter 500nm-1500nm, preferred 700-1200nm；It is saturated magnetic
Change intensity and be not less than 40 emu/g, is preferably not less than 50 emu/g.The every mg of nano biological magnetic bead is obtained in combination with being not less than
The DNA of 10ug especially can achieve every mg in combination with the DNA for being not less than 15ug.
3, the method for the invention makes its coordinative role by improving to raw material, conditional parameter and method and step,
By surface modification and further functionalization, the ferromagnetism of Fe3O4 magnetic core is controlled and optimizes, while enhancing nano biological magnetic bead
Dispersion stabilization and biocompatibility.It is extracted using the nano biological magnetic bead of the method for the present invention preparation, better quality can be obtained
Nucleic acid, lay the foundation for follow-up study.
Detailed description of the invention
Fig. 1 is the droplet measurement report figure of the nano biological magnetic bead prepared using embodiment 1；
Fig. 2 is the droplet measurement report figure of the nano biological magnetic bead prepared using embodiment 2；
Fig. 3 is that the amplification of the fluorogenic quantitative detection of the DNA/RNA sample extracted using nano biological magnetic bead prepared by the present invention is bent
Line chart；
Fig. 4 is the fluorogenic quantitative detection result figure based on Fig. 3.
Specific embodiment
A kind of preparation method of the nano biological magnetic bead for ultramicron nucleic acid extraction, it includes following steps:
Step 1: nano ferriferrous oxide dispersion liquid preparation
It takes 100g~10000g nano ferriferrous oxide powder by multiple ethanol washing, then uses again and contain 1% surfactant
Milli-Q water for several times, be finally dispersed in 5~10L and contain in the ethanol water of a certain amount of oleic acid, obtain nanometer four oxidation
Three-iron primary solution.Wherein the content of the oleic acid is 1~100ml, preferably 5~50ml.
Above-mentioned nano ferriferrous oxide primary solution is handled with high pressure homogenizer, high pressure homogenizer running parameter 80MPa
~150MPa, homogeneous processing 1~6 time, obtain nano ferriferrous oxide dispersion liquid.
Step 2: silica is wrapped up on nano ferriferrous oxide surface
The nano ferriferrous oxide dispersion liquid that above-mentioned steps one obtain is put into reaction kettle, be added comprising tetraethyl orthosilicate and
100~2000ml of mixed liquor of ammonium hydroxide reacts 1~5h, is washed 2 times with alcohol solution, then with the water containing surfactant
Solution washing, is finally dispersed in the aqueous solution containing surfactant, obtains magnetic bead solution
Above-mentioned gained magnetic bead solution high pressure homogenizer is handled 1~2 time, high pressure homogenizer 60~120MPa of running parameter, place
Continue ultrasonic agitation 12~for 24 hours after reason, control temperature is no more than 30 DEG C during ultrasonic agitation, then with milli-Q water to molten
Liquid PH is in neutrality, and is stored in ultrapure water to get the nano biological magnetic bead for ultramicron nucleic acid extraction.
The nano ferriferrous oxide powder can be to be made by Hydrolyze method, hydro-thermal method, coprecipitation or ball-milling method
, preferably coprecipitation, ball-milling method.
Surfactant described in step 1 is that Qula is led to, nonylphenol polyoxyethylene ether, vegetable oil (are with maize germ oil
Well), the mixture of one or more of citric acid, dodecyl sodium sulfate, cetyl trimethylammonium bromide.
The nano ferriferrous oxide powder primary particle size is 10nm-500nm, preferably 50-300nm；Saturation magnetization
Not less than 50 emu/g, preferably not less than 60 emu/g.
After nano ferriferrous oxide described in step 1 settles completely in 1% aqueous surfactant solution, solid-to-liquid ratio
Higher than 1:1, preferably no greater than 1:3.
A kind of high pressure homogenizer running parameter of step is 80MPa~150MPa, preferably 100MPa~120MPa；Processing
Number is 1~6 time, preferably 3~5 times.
Nano ferriferrous oxide primary solution described in step 1 before being handled with high pressure homogenizer, processing after, treatment process
In be in suspension dispersity, be at the state can with but be not limited to utilize stirring, one of ultrasound or a variety of sides
Formula shares；Ultrasonic power described in step 2 is not less than 800W, preferably not less than 1200W.
The ratio of tetraethyl orthosilicate and ammonium hydroxide is in mixed liquor comprising tetraethyl orthosilicate and ammonium hydroxide in step 2
0.2-1.0, preferably 0.3~0.6；The additional amount of mixed liquor comprising tetraethyl orthosilicate and ammonium hydroxide is 100~2000ml, preferably
150~1000ml.The reaction time is 1~5h, preferably 2~4h after addition.
Alcohol is the compound of one or more of ethyl alcohol, isopropanol, glycerine, institute in alcohol solution described in step 2
The concentration of alcohol in alcohol solution is stated not less than 50%, preferably not less than 80%.
The volume fraction of surfactant is 1%~5% in aqueous solution described in step 2 containing surfactant, described
Surfactant is that Qula is led to, in polyethylene glycol, glycerol, citric acid, dodecyl sodium sulfate, cetyl trimethylammonium bromide
One or more of mixtures.
High pressure homogenizer running parameter described in step 2 is 60~120MPa, preferably 60~90MPa.
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described.
Embodiment 1
A kind of preparation method of the nano biological magnetic bead for ultramicron nucleic acid extraction, comprising the following specific steps
Step 1, nanometer Fe_3O_4 dispersion solution preparation
It takes 300g nano ferriferrous oxide powder ethanol washing 5 times, is then washed 5 times with 1% citric acid solution again, final point
It is dispersed in 5L to contain in 95% ethanol solution of 10ml oleic acid, obtains nano ferriferrous oxide primary solution.
Above-mentioned nano ferriferrous oxide primary solution high pressure homogenizer is handled 3 times, high pressure homogenizer running parameter
100MPa obtains nano ferriferrous oxide dispersion liquid.
Step 2: wrapping up silica on nano ferriferrous oxide surface
Above-mentioned nano ferriferrous oxide dispersion liquid is put into reaction kettle, ammonium hydroxide 600ml is added, tetraethyl orthosilicate is added dropwise
300ml, react 3h, with 60% isopropanol water wash 1 time, then with 70% ethanol washing 1 time, then with contain 1% aqueous citric acid solution
Washing 3 times, is finally dispersed in the aqueous solution containing 1% citric acid.
Above-mentioned magnetic bead solution high pressure homogenizer is handled 2 times, homogenizer running parameter 60MPa, continues ultrasound after processing
12h, ultrasonic power 800W are stirred, control temperature is no more than 30 DEG C.Then it is in neutrality, is stored in milli-Q water to solution PH
To get the nano biological magnetic bead for ultramicron nucleic acid extraction in ultrapure water.
Quality evaluation is carried out to the nano biological magnetic bead using above method preparation, magnetic bead particle size data (appraisal report) is shown in
Fig. 1；As shown in Figure 1, uniform particle sizes, average grain diameter is in 500nm-1500nm, preferable 700-1200nm.Magnetic bead surfaces hydroxyl contains
Amount and maximum DNA binding capacity determination data see attached list 1；
Embodiment 2
A kind of preparation method of the nano biological magnetic bead for ultramicron nucleic acid extraction, comprising the following specific steps
Step 1, nanometer Fe_3O_4 dispersion solution preparation
With embodiment one
Step 2: wrapping up silica on nano ferriferrous oxide surface
Above-mentioned nano ferriferrous oxide dispersion liquid is put into reaction kettle, ammonium hydroxide 500ml is added, tetraethyl orthosilicate is added dropwise
200ml reacts 3h, then with 70% ethanol washing 2 times, then with the aqueous solution containing 1% dodecyl sodium sulfate washs 3 times, most
It is dispersed in the aqueous solution containing 1% citric acid afterwards.
Above-mentioned magnetic bead solution high pressure homogenizer is handled 2 times, homogenizer running parameter 80MPa, continues ultrasound after processing
18h, ultrasonic power 800W are stirred, control temperature is no more than 30 DEG C.Then it is in neutrality, is stored in milli-Q water to solution PH
To get the nano biological magnetic bead for ultramicron nucleic acid extraction in ultrapure water.To the nano biological using above method preparation
Magnetic bead carries out quality evaluation, and magnetic bead particle size data (appraisal report) is shown in Fig. 1；As shown in Figure 1, uniform particle sizes.Magnetic bead surfaces hydroxyl
Content and maximum DNA binding capacity determination data see attached list 1；
Embodiment 3
A kind of preparation method of the nano biological magnetic bead for ultramicron nucleic acid extraction, comprising the following specific steps
Step 1, nanometer Fe_3O_4 dispersion solution preparation
It takes 500g nano ferriferrous oxide powder ethanol washing 5 times, then washs 5 with 1% sodium dodecyl sulfate solution again
It is secondary, it is finally dispersed in 5L and contains in 90% ethanol solution of 20ml oleic acid, obtain nano ferriferrous oxide primary solution.
Above-mentioned nano ferriferrous oxide primary solution high pressure homogenizer is handled 4 times, high pressure homogenizer running parameter is
120MPa obtains nano ferriferrous oxide dispersion liquid.
Step 2: wrapping up silica on nano ferriferrous oxide surface
One gained nano ferriferrous oxide dispersion liquid of above-mentioned steps is put into reaction kettle, ammonium hydroxide 500ml is added, positive silicic acid is added dropwise
Tetra-ethyl ester 200ml reacts 4h, then with 80% ethanol washing 2 times, then with the aqueous solution containing 1% dodecyl sodium sulfate washs 3
It is secondary, it is finally dispersed in the aqueous solution containing 1% citric acid.
Above-mentioned magnetic bead solution high pressure homogenizer is handled 2 times, homogenizer running parameter 100MPa, continues ultrasound after processing
For 24 hours, ultrasonic power 1200W, control temperature is no more than 30 DEG C for stirring.Then it is in neutrality, is saved with milli-Q water to solution PH
To get the nano biological magnetic bead for ultramicron nucleic acid extraction in ultrapure water.Magnetic bead surfaces hydroxy radical content and maximum DNA knot
Resultant determination data sees attached list 1；
Table 1: the nano biological magnetic bead surfaces hydroxy radical content and maximum DNA binding capacity of embodiment 1-3 preparation
| Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| Hydroxy radical content | 0.37mmol/g | 0.4mmol/g | 0.36mmol/g |
| DNA binding capacity | 18ug/mg | 19ug/mg | 18ug/mg |
Embodiment 4
A kind of nano biological magnetic bead for ultramicron nucleic acid extraction prepared using preparation method of the present invention, is specifically made
It is as follows with (nucleic acid extraction):
(1) 20 μ l lysate B are added in 1.5 ml EP pipes；
(2) 200 μ l Method for Microarray Applications are added；
(3) be added 300 μ l lysate AS, vortex oscillation be uniformly mixed, 56 DEG C water-bath 10 minutes.
(4) EP pipe is taken out from incubation equipment, 10 μ l of above-mentioned example magnetic bead is added, 300 μ l of isopropanol, room is added
Temperature is mixed by inversion 5 minutes.Then EP pipe is placed on magnetic frame and carries out Magneto separate, stood to magnetic bead and be all adsorbed to tube wall, inhaled
Abandon liquid in pipe.
(5) magnetic frame is removed, 500 μ l cleaning solution AW are added, turns upside down and shakes EP pipe 2 minutes.
(6) EP pipe is placed on magnetic frame, stands to magnetic bead and is all adsorbed to tube wall, inhaled and abandon liquid in pipe.
(7) magnetic frame is removed, 500 μ l cleaning solution SW are added, turns upside down and shakes EP pipe 2 minutes.
(8) EP pipe is placed on magnetic frame, stands to magnetic bead and is all adsorbed to tube wall, inhaled and abandon liquid in pipe.
(9) pipe lid is opened, magnetic bead is allowed to dry in safety cabinet 5 minutes.
(10) magnetic frame is removed, 50-100 μ l eluent S(nuclease-free is added) or deionized water, flick EP pipe
Make magnetic bead all infiltration in a liquid, 56 DEG C water-bath 5-10 minute, during which every 2-3 minutes jog EP pipes mixing magnetic bead.
(11) EP pipe is placed on magnetic frame, stands to magnetic bead and is all adsorbed to tube wall, draw supernatant to a new EP pipe
(sterile no RNase pollution) is to get the DNA/RNA for arriving sample.
(12) the DNA/RNA sample for taking 20ul to obtain carries out fluorogenic quantitative detection, and testing result is as shown in attached drawing 3.
From the figure 3, it may be seen that the nano biological magnetic bead that the present invention obtains is for the available better quality of ultramicron nucleic acid extraction
Nucleic acid, expanded by qPCR, the yield of nucleic acid is stablized, and threshold value is forward.
It should be noted that embodiment described above is interpreted as illustrative, to be not intended to limit the present invention protection
Range, protection scope of the present invention are subject to claims.To those skilled in the art, without departing substantially from of the invention real
Under the premise of matter and range, some nonessential modifications and adaptations made to the present invention still fall within protection scope of the present invention.
Claims (10)
1. a kind of preparation method of the nano biological magnetic bead for ultramicron nucleic acid extraction, it is characterised in that: it includes following steps
It is rapid:
Step 1: nano ferriferrous oxide dispersion liquid preparation
100g~10000g nano ferriferrous oxide powder is taken to be washed again with the aqueous solution containing surfactant after ethanol washing
It washs, is then dispersed in 5~10L and contains in the ethanol water of oleic acid, obtain nano ferriferrous oxide primary solution；By four oxygen of nanometer
Change three-iron primary solution to be handled with high pressure homogenizer, the high pressure homogenizer running parameter is 80MPa~150MPa, number is handled
It is 1~6 time, obtains nano ferriferrous oxide dispersion liquid；
Step 2: silica is wrapped up on nano ferriferrous oxide surface
Nano ferriferrous oxide dispersion liquid obtained by step 1 is put into reaction kettle, being added includes tetraethyl orthosilicate and ammonium hydroxide
100~2000ml of mixed liquor reacts 1~5h, is washed again with the aqueous solution containing surfactant after being washed with alcohol solution, most
It is dispersed in the aqueous solution containing surfactant afterwards, obtains magnetic bead solution；By gained magnetic bead solution with high pressure homogenizer handle 1~
2 times, the high pressure homogenizer running parameter is 60~120MPa, and after treatment continues ultrasonic agitation 12~for 24 hours, ultrasonic agitation
In the process control temperature be no more than 30 DEG C, be then in neutrality with milli-Q water to solution PH, be stored in ultrapure water to get with
In the nano biological magnetic bead of ultramicron nucleic acid extraction.
2. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 1, special
Sign is: the nano ferriferrous oxide powder is as made from Hydrolyze method, hydro-thermal method, coprecipitation or ball-milling method.
3. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 1, special
Sign is: the volume fraction of surfactant is 1% in the aqueous solution described in step 1 containing surfactant；The surface
Activating agent is logical Qula, nonylphenol polyoxyethylene ether, vegetable oil, citric acid, dodecyl sodium sulfate and cetyl trimethyl
The mixture of one or more of ammonium bromide.
4. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 3, special
Sign is: the vegetable oil is maize germ oil.
5. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 1, special
Sign is: the volume fraction of surfactant is 1%-5% in the aqueous solution described in step 2 containing surfactant；The table
Face activating agent is that Qula is led to, in polyethylene glycol, glycerol, citric acid, dodecyl sodium sulfate and cetyl trimethylammonium bromide
One or more of mixtures.
6. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 1, special
Sign is: the partial size of nano ferriferrous oxide powder described in step 1 is 10nm-500nm, and saturation magnetization is not less than 50
emu/g。
7. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 1, special
Sign is: the content of oleic acid is 1~100ml in the ethanol water containing oleic acid.
8. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 1, special
Sign is: ultrasonic power described in step 2 is not less than 800W.
9. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 1, special
Sign is: the ratio of tetraethyl orthosilicate and ammonium hydroxide is in the mixed liquor comprising tetraethyl orthosilicate and ammonium hydroxide described in step 2
0.2~1.0.
10. a kind of preparation method of nano biological magnetic bead for ultramicron nucleic acid extraction according to claim 1, special
Sign is: alcohol is the compound of one or more of ethyl alcohol, isopropanol, glycerine in alcohol solution described in step 2；Institute
The concentration of alcohol in alcohol solution is stated not less than 50%.
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