CN107402209A - It is a kind of to be used to detect test strips of LDL-C and preparation method thereof in serum - Google Patents
It is a kind of to be used to detect test strips of LDL-C and preparation method thereof in serum Download PDFInfo
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Abstract
The present invention relates to a kind of test strips for being used to detect LDL-C in serum, test strips include dry chemical reaction plate, dry chemical reaction plate is by the way that reagent pad is immersed in chemical reagent, then it is made through drying, chemical reagent is using preferred concentration and cholesterol esterase, cholesterol oxidase, peroxidase, metal salt, developer, APEO and the SYNPERONIC PE/F68 of ratio etc..The equipment cost of the present invention is low, easily prepared acquisition, and rapid reaction, easy to operate.
Description
Technical field
It is used to detect test strips of LDL-C and preparation method thereof in serum the present invention relates to a kind of.
Background technology
Abnormalities of sugar/lipid metabolism is one of coronary heart disease (CHD) most important hazards.Many larger scale clinical researchs have been filled
Dividing proves cholesterol particularly important function of the LDL-C (LDL-C) in the occurrence and development of coronary heart disease, drop
Low LDL-C can substantially reduce coronary event.LDL-C rise is proportionate with Incidence of CHD, is hat
One of important analysis index of worry.Traditional LDL-C concentration is obtained by way of separation determination or indirect approximate calculation.Point
Lipoprotein composition in blood sample is pressed into HDL, low density lipoprotein by certain step using ultracentrifuge from measure
After the classification such as albumen and chylomicron is separated, then individually it is measured.This method is high to equipment requirement, and time-consuming,
Complex operation.And the mode of approximate calculation is consolidated according to T-CHOL (TC), triglycerides (TG), HDL courage indirectly
LDL-C approximation is calculated by Friedewald formula for alcohol (HDL-C) numerical value, but this method is only applicable to TG<4.52
mmol/L.When CM and TG higher in serum be present, calculated value deviation is larger, and many factors also influence the accuracy of formula.Cause
This, only Direct Determination LDL-C can provide accurate diagnosis basis for clinic.Many commercialized products exist in recent years
LDL-c direct detection is realized on full automatic biochemical apparatus.The realization of the program mainly passes through some special surface-actives
Agent, in the enzyme system of traditional T-CHOL detection reaction, HDL is protected and is allowed to be not involved in reacting, or delay HDL portions
The reaction speed divided, from the target for reaching only LDL participation enzyme system reactions, finally only obtain the concentration inspection of cholesterol in LDL
Survey result, i.e., the LDL-c concentration in sample.
So far, LDL-c method is mainly direct assay and null method two in direct method detection blood serum sample
Kind, the concrete technical scheme of both approaches is as described below：
1st, direct assay：Refer to suppress in blood serum sample using materials such as a- cyclodextrin, dextran sulfate and Brij58
The non-LDL-C lipoprotein such as HDL, VLDL and cholesterol esterase and cholesterol oxidase reaction (can also claim selective depression),
So as to only make LDL-C by above two enzyme hydrolysis and method for measuring.
Using this method usually using double reagent reaction system.The sodium sulphate of cyclodextrin containing a- in reagent 1, dextran sulfate,
MgCl2, EMSE and PBS.Contain CHER, CHOD peroxidase, 4-AA, surfactant and PBS in reagent 2.A- cyclodextrin sulphur
Sour sodium is in a small amount of DS and Mg2+In the presence of, chylomicron (CM) and the cholesterol in VLDL components and the reaction of enzymatic reagent can be reduced,
Special surface activating agent can reduce the reaction of HDLC component cholesterol, and both combine and then optionally determine LDL-c.
, null method：It is that non-LDL-c and LDL-c points of two one-step hydrolysis are made with the double reagent of different surfactants, because first disappearing
Consume non-LDL-c and be referred to as null method, be to apply wider direct measuring method at present.
This method equally uses double reagent reaction system.Mainly there are surfactant null method, catalase null method
Deng.The reagent of Japanese first chemical Genxyme Diagnostics companies belongs to such method, is that use at present is most extensive both at home and abroad
A kind of reagent.Surfactant 1 in its reagent 1 can change HDL, CM and the VLDL structure beyond LDL-c and dissociate, and be released
The micronized cholesterol molecule released and cholesterol enzyme reagent reacting, caused H2O2It is consumed when lacking coupling molecule without aobvious
Color, now LDL-c particles dissociation release cholesterol, participates in enzyme reaction and develops the color, the color and luster depth is proportional to LDL-c amounts.
Both the above detection method belongs to bi-component biochemical reagents, is required to spectrophotometer or full-automatic biochemical analysis
Instrument performs correlated response, and carries out result and calculate analysis.
Therefore, when LDL-c concentration is detected in above-mentioned technology, there are the following problems：Using supercentrifugation separation LDL with
HDL scheme is, it is necessary to using expensive Ultracentrifuge as main separation equipment, and experimental procedure is more, behaviour
Make sufficiently complex, it is necessary to prepare plurality of reagents, it is most important that quite time-consuming, whole disengaging time was often beyond 6 hours.It is and straight
Connecing detection method scheme then needs large automatic equipment as full automatic biochemical apparatus, with high costs.
Authorization Notice No. is CN101864475B, and authorized announcement date is that 2013-11-6 measurement low-density lipoprotein courage is consolidated
The method of alcohol, it discloses the dry analysis element for measuring LDL-c, its preparation method is to be coated with gelatin, thickness below
Spend the colourless, transparent of polyethylene terephthalate for 180 microns, aqueous gelatin solution is coated with smooth film, and dry, extremely
Dried thickness reaches 14 microns, the aqueous solution of the coating with consisting of, next dries.4-AA
0.32g/m2、TOOS 0.62 g/m2, peroxidase 12.75KU/ m2.On the above-mentioned films with about 30 m2Amount each
Face Jia Shui makes its moistening.Then gently apply pressure, be laminated and formed with No. 36 specification braidings equivalent to the Direct-spinning of PET Fiber of 50 Neils
Light knitted or woven fabrics, and dry.Then, the aqueous solution of following compositions is coated with above-mentioned cloth, is next dried.MOPS
(pH7.0) 1.67 g/m2, the g/m of polyglycereol tri-styryl phenyl ether 5.022、Pluronic L 121 39.75 g/m2、
Cholesterol esterase（Lipoprotein lipase, Toyobo） 0.65 KU/ m2, cholesterol oxidase（Recombination bacillus coli,
Kikkoman） 0.13 KU/ m2.It can be seen that dry analysis element made of the patent is to need two kinds of reagents to be respectively fixed to not
On same film, therefore, preparation method is cumbersome.
The content of the invention
The technical problems to be solved by the invention be to provide a kind of cost it is cheap, prepare be simply to detect serum in it is low
The test strips of density lipoprotein-cholesterol.
Another technical problem to be solved by this invention is to provide the preparation method of above-mentioned test strips.
To solve above technical problem, the present invention adopts the following technical scheme that：
It is described it is an object of the present invention to provide a kind of test strips for being used to detect LDL-C in serum
Test strips include dry chemical reaction plate, and described dry chemical reaction plate is by the way that reagent pad is immersed in chemical reagent, Ran Houjing
Dry and be made, described chemical reagent includes cholesterol esterase, the 20 ~ 80KU/L cholesterol oxidation that concentration is 1 ~ 30KU/L
Enzyme, 250 ~ 350mM metal salt, 100 ~ 400KU/L horseradish peroxidase, 15 ~ 25mM 4-AA, 40 ~
60 mM developer, 0.12 ~ 0.2g/mL polyethenoxy ether class surfactant, 1.5 ~ 2.5g/L oxirane-epoxy
Propane copolymer surfactants and phosphate buffer.
Preferably, described chemical reagent includes cholesterol esterase, the 40 ~ 60KU/L cholesterol that concentration is 5 ~ 15KU/L
Oxidizing ferment, 280 ~ 320mM metal salt, 150 ~ 250KU/L horseradish peroxidase, 18 ~ 22mM 4-AA,
45 ~ 55mM developer, 0.14 ~ 0.18g/mL polyethenoxy ether class surfactant, 1.8 ~ 2.2g/L oxirane-ring
Ethylene Oxide copolymer surfactants and phosphate buffer.
It is further preferred that it is 5 ~ 8KU/L cholesterol esterase, 40 ~ 45KU/L that described chemical reagent, which includes concentration,
Cholesterol oxidase, 280 ~ 290mM metal salt, 150 ~ 180KU/L horseradish peroxidase, 18 ~ 19mM 4- amino peace
For than woods, 45 ~ 48mM developer, 0.14 ~ 0.15g/mL polyethenoxy ether class surfactant, 1.8 ~ 1.9g/L epoxy
Oxide-propylene oxide copolymer surfactants and phosphate buffer.
Preferably, described metal salt is selected from magnesium sulfate, magnesium chloride, sodium sulphate, sodium chloride, calcium chloride, magnesium phosphate, phosphorus
One or more in sour sodium；More preferably magnesium sulfate or magnesium chloride.
Preferably, described developer is selected from N- (2- hydroxyl -3- sulfopropyls) -3 5- dimethoxyaniline sodium salts
（HDAOS）, N- ethyls-N- (the third sulfo groups of 2- hydroxyls -3-) meta-aminotoluene（TOOS）, N, N- bis- (4- sulfopropyls) -3,5- dimethyl
Aniline sodium salt（MADB）, N- ethyls-N- (2- hydroxyl -3- sulfopropyls) -3,5- dimethylaniline sodium salt monohydrates（MAOS）、N-
Ethyl-N- (3- sulfopropyls) -3- aminoanisole sodium salts（ADPS）, TODB
（TODB）, N- ethyls-N- (3- sulfopropyls) -3- methylaniline sodium salts（TOPS）, N- ethyls-N- (2- hydroxyl -3- sulfopropyls) -
3,5- dimethoxyaniline sodium salts（DAOS）, NBT（NBT）, p-Iodonitrotetrazolium violet it is blue（INT）, four nitrogen
Azoles is blue（BT）In one or more；More preferably N- ethyls-N- (the third sulfo groups of 2- hydroxyls -3-) meta-aminotoluene（TOOS）.
Preferably, described polyethenoxy ether class surfactant is selected from AEO, polyoxyethylene nonyl phenyl
One or more in vinethene, sorbitan monooleate APEO, polyoxyethylene laurel ether.
It is further preferred that described AEO is in AEO-3, AEO-5, AEO-7, AEO-10
It is one or more.
It is further preferred that described NPE is one kind or more in NP-4, NP-10, NP-15
Kind.
It is further preferred that described sorbitan monooleate APEO is tween-20 and/or tween-
80。
It is further preferred that described polyoxyethylene laurel ether is Brij-35 and/or Brij-58.
Preferably, the number-average molecular weight of described PEP-101 surfactant be 5000 ~
15000。
It is further preferred that the number-average molecular weight of described PEP-101 surfactant is
6000~10000。
It is further preferable that the number-average molecular weight of described PEP-101 surfactant be 8000 ~
9000。
Preferably, described PEP-101 surfactant is selected from poloxamer F124, pool Lip river
One or more in husky nurse F88, Plurinic L-122, Plurinic L-101.
Preferably, the material of described reagent pad is nitrocellulose filter, glass fibre element film, polyvinylidene fluoride element
One kind in film, poly-vinegar acid esters cellulose membrane, Polysulfonamide, polyethyleneimine film.More preferably poly- acetic ester fiber
Plain film.
Preferably, the aperture of the reagent pad be 0.3 ~ 0.5 micron, more preferably 0.35 ~ 0.45 micron, most preferably
For 0.4 micron.Preferably, described test strips also include sample release pad of the lining in described dry chemical reaction plate.
It is further preferred that the material of described sample release pad is glass fiber membrane.
It is further preferred that the thickness of the sample release pad is 1 ~ 3 millimeter, more preferably 1.5 ~ 2.5 millimeters, most preferably
For 2 millimeters.
Described it is used to detect LDL-C in serum it is a further object to provide a kind of
The preparation method of test strips, comprises the following steps：
Step（1）, by described cholesterol esterase, described cholesterol oxidase, described metal salt, described horseradish peroxide
Compound enzyme, described 4-AA, described developer and described phosphate buffer are mixed to form mixed liquor；
Step（2）, add into described mixed liquor described polyethenoxy ether class surfactant and described oxirane-
Epoxy propane copolymer surfactant, it is mixed to form described chemical reagent；
Step（3）, described reagent pad is put into described chemical reagent, soak 60 ~ 90min, then done at 45 ~ 50 DEG C
Dry 40 ~ 60min, described described dry chemical reaction plate is made.
Preferably, described preparation method also includes described sample discharging pad in described dry chemical reaction plate
The step of.
Due to the implementation of above technical scheme, the present invention has the following advantages that compared with prior art：
The invention provides a kind of test strip of LDL-C in serum based on dry chemical method, and it is logical
Cross to dry after reagent pad is immersed in chemical reagent and be made, be carried on different films, make respectively without using two kinds of reagents
Preparation Method is simple, and the testing result of the low and obtained test strips of cost is accurate, and rapid reaction is easy to operate.
Brief description of the drawings
Accompanying drawing 1 is the phase of the testing result and full automatic biochemical apparatus testing result of the LDL-c dry chemical reaction plates of embodiment 1
Guan Xingtu；
Accompanying drawing 2 is the correlation of the testing result and full automatic biochemical apparatus testing result of the LDL-c dry chemical reaction plates of embodiment 2
Figure；
Accompanying drawing 3 is the correlation of the testing result and full automatic biochemical apparatus testing result of the LDL-c dry chemical reaction plates of embodiment 3
Figure.
Embodiment
With reference to specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following implementation
Example.The implementation condition used in embodiment can do further adjustment, unreceipted implementation according to specifically used different requirements
Condition is the normal condition in the industry.What those of ordinary skill in the art were obtained under the premise of creative work is not made
All other embodiment, belongs to the scope of protection of the invention.
The commercially available acquisition of agents useful for same of the present invention.
Embodiment 1：
(1）Prepare T-CHOL（TC）Detection reagent：By cholesteryl esterase（CHER）5KU/L, cholesterol oxidase（CHOD）
40KU/L, MgSO4280mM, horseradish peroxidase 150KU/L, 4-AA 18 mM, N- ethyl-N- (2- hydroxyls
The sulfo groups of base -3- third) meta-aminotoluene（TOOS）45 mM concentration, with phosphoric acid（PBS）For buffer solution, above reagent is all added
In 15mL EP pipes, it is well mixed.
）In the reagent that the first step obtains, 0.14g Tween-20s are added by 1mL reagents（Tween-20）, then by 1.8g/L
Concentration add number-average molecular weight be 8400 poloxamer F124, be well mixed.
）Aperture 0.4um nitrocellulose filter is cut into the wide strips of about 0.5cm, is put into the solution that previous step prepares
In, soak 1.5 hours.Then take out, be put into electric drying oven with forced convection, 60min is dried under 45 degrees Celsius.The film dried completely
Bar is LDL-c dry chemical reaction plates.
）The glass fiber membrane thick using 2mm is served as a contrast and is made on LDL-c dry chemical reaction plates as sample release pad
Test strips.
（5）Test serum is added dropwise in sample release pad with liquid-transfering gun, serum is by sample release pad uniformly by LDL-c
Dry chemical reaction plate is impregnated with, and changes colour with reaction, realizes LDL-c dry chemical detection.
（6）Use the test strips of the present embodiment, the reagent using Japanese first chemical Genxyme Diagnostics companies
LDL-c detection is carried out to clinical serum sample respectively by automatic clinical chemistry analyzer, as a result as Fig. 1, Fig. 1 do for LDL-c
The testing result of piece that chemically reacts and the correlation data of full automatic biochemical apparatus testing result, it is known that this LDL-c dry chemicals are reacted
The testing result of piece has preferable correlation with full automatic biochemical apparatus testing result.
Embodiment 2：
(1）Prepare T-CHOL（TC）Detection reagent：By cholesteryl esterase（CHER）15KU/L, cholesterol oxidase（CHOD）
60KU/L, MgSO4320mM, horseradish peroxidase 250KU/L, 4-AA 22 mM, N- ethyl-N- (3-
Sulfopropyl) -3- methylaniline sodium salts（TOPS）55 mM concentration, with phosphoric acid（PBS）For buffer solution, above reagent is all added
In the EP pipes for entering 15mL, it is well mixed.
）In the reagent that the first step obtains, 0.18g Tween-20s are added by 1mL reagents（Tween-20）, then by 2.2g/L
Concentration add number-average molecular weight be 8400 poloxamer F88, be well mixed.
）Aperture 0.4um nitrocellulose filter is cut into the wide strips of about 0.5cm, is put into the solution that previous step prepares
In, soak 1.5 hours.Then take out, be put into electric drying oven with forced convection, 60min is dried under 45 degrees Celsius.The film dried completely
Bar is LDL-c dry chemical reaction plates.
）The glass fiber membrane thick using 2mm is served as a contrast and is made on LDL-c dry chemical reaction plates as sample release pad
Test strips.
（5）Test serum is added dropwise in sample release pad with liquid-transfering gun, serum is by sample release pad uniformly by LDL-c
Dry chemical reaction plate is impregnated with, and changes colour with reaction, realizes LDL-c dry chemical detection.
（6）Using the test strips of the present embodiment and using Japanese first chemical Genxyme Diagnostics companies
Reagent carries out LDL-c detection by automatic clinical chemistry analyzer to five parts of clinical serum samples respectively, as a result as Fig. 2, Fig. 2 are
The testing result of LDL-c dry chemical reaction plates and the correlation data of full automatic biochemical apparatus testing result, it is known that this LDL-c desiccation
The testing result for learning reaction plate has preferable correlation with full automatic biochemical apparatus testing result.
Embodiment 3：
(1）Prepare T-CHOL（TC）Detection reagent：By cholesteryl esterase（CHER）10KU/L, cholesterol oxidase（CHOD）
50KU/L, MgCl2300mM, horseradish peroxidase 200KU/L, the mM of 4-AA 20, chlorination nitro tetrazole
It is blue（NBT）50 mM concentration, with phosphoric acid（PBS）For buffer solution, above reagent is all added in 15mL EP pipes, mixing is equal
It is even.
）In the reagent that the first step obtains, 0.16g NP-10 are added by 1mL reagents, then it is equal by 2g/L concentration addition number
Molecular weight is 6000 poloxamer F88, is well mixed.
）Aperture 0.4um nitrocellulose filter is cut into the wide strips of about 0.5cm, is put into the solution that previous step prepares
In, soak 1.5 hours.Then take out, be put into electric drying oven with forced convection, 60min is dried under 45 degrees Celsius.The film dried completely
Bar is LDL-c dry chemical reaction plates.
）The glass fiber membrane thick using 2mm is served as a contrast and is made on LDL-c dry chemical reaction plates as sample release pad
Test strips.
（5）Test serum is added dropwise in sample release pad with liquid-transfering gun, serum is by sample release pad uniformly by LDL-c
Dry chemical reaction plate is impregnated with, and changes colour with reaction, realizes LDL-c dry chemical detection.
（6）Use the test strips of the present embodiment, the reagent using Japanese first chemical Genxyme Diagnostics companies
LDL-c detection is carried out to clinical serum sample respectively by automatic clinical chemistry analyzer, as a result as shown in figure 3, as can be seen from Figure 3
The testing result of this LDL-c dry chemical reaction plates has preferable correlation with full automatic biochemical apparatus testing result.
The present invention is described in detail above, its object is to allow the personage for being familiar with this art to understand this
The content of invention is simultaneously carried out, and it is not intended to limit the scope of the present invention, all Spirit Essence institutes according to the present invention
The equivalent change or modification of work, it should all cover within the scope of the present invention.
Claims (10)
- A kind of 1. test strips for being used to detect LDL-C in serum, it is characterised in that：Described test strips bag Dry chemical reaction plate is included, then described dry chemical reaction plate is made by the way that reagent pad is immersed in chemical reagent through drying, Described chemical reagent include concentration be 1 ~ 30KU/L cholesterol esterase, 20 ~ 80KU/L cholesterol oxidase, 250 ~ 350mM metal salt, 100 ~ 400KU/L horseradish peroxidase, 15 ~ 25mM 4-AA, 40 ~ 60 mM The epoxy ethane-epoxy propane copolymerization of developer, 0.12 ~ 0.2g/mL polyethenoxy ether class surfactant, 1.5 ~ 2.5g/L Thing surfactant and phosphate buffer.
- 2. the test strips according to claim 1 for being used to detect LDL-C in serum, it is characterised in that： Described chemical reagent include concentration be 5 ~ 15KU/L cholesterol esterase, 40 ~ 60KU/L cholesterol oxidase, 280 ~ 320mM metal salt, 150 ~ 250KU/L horseradish peroxidase, 18 ~ 22mM 4-AA, 45 ~ 55mM it is aobvious The epoxy ethane-epoxy propane copolymerization of toner, 0.14 ~ 0.18g/mL polyethenoxy ether class surfactant, 1.8 ~ 2.2g/L Thing surfactant and phosphate buffer.
- 3. the test strips according to claim 1 or 2 for being used to detect LDL-C in serum, its feature exist In：Described metal salt is one kind in magnesium sulfate, magnesium chloride, sodium sulphate, sodium chloride, calcium chloride, magnesium phosphate, sodium phosphate It is or a variety of；Described developer is selected from N- (2- hydroxyl -3- sulfopropyls) -3 5- dimethoxyanilines sodium salts, N- ethyls-N- (2- hydroxyls The sulfo groups of base -3- third) meta-aminotoluene, (4- the sulfopropyls) -3,5- dimethylanilines of N, N- bis- sodium salt, N- ethyls-N- (2- hydroxyls -3- Sulfopropyl) -3,5- dimethylaniline sodium salts monohydrate, N- ethyls-N- (3- sulfopropyls) -3- aminoanisoles sodium salt, N, N- Two (4- sulphur butyls) -3- methylanilines disodium salts, N- ethyls-N- (3- sulfopropyls) -3- methylanilines sodium salt, N- ethyls-N- (2- hydroxyl -3- sulfopropyls) -3,5- dimethoxyanilines sodium salt, NBT, p-Iodonitrotetrazolium violet indigo plant, four One or more in nitrogen azoles basket；Described polyethenoxy ether class surfactant is selected from AEO, NPE, dehydration One or more in sorbitol monooleate APEO, polyoxyethylene laurel ether.
- 4. the test strips according to claim 1 or 2 for being used to detect LDL-C in serum, its feature exist In：The number-average molecular weight of described PEP-101 surfactant is 5000 ~ 15000.
- 5. the test strips according to claim 4 for being used to detect LDL-C in serum, it is characterised in that： The number-average molecular weight of described PEP-101 surfactant is 6000 ~ 10000.
- 6. the test strips according to claim 1 or 2 for being used to detect LDL-C in serum, its feature exist In：The material of described reagent pad is nitrocellulose filter, glass fibre element film, polyvinylidene fluoride element film, poly-vinegar acid esters One kind in cellulose membrane, Polysulfonamide, polyethyleneimine film.
- 7. the test strips according to claim 1 or 2 for being used to detect LDL-C in serum, its feature exist In：Described test strips also include sample release pad of the lining in described dry chemical reaction plate.
- 8. the test strips according to claim 7 for being used to detect LDL-C in serum, it is characterised in that： The material of described sample release pad is glass fiber membrane, and the thickness of the glass fiber membrane is 1 ~ 3 millimeter.
- A kind of 9. test paper for being used to detect LDL-C in serum as any one of claim 1 to 8 The preparation method of bar, it is characterised in that：Comprise the following steps：Step（1）, by described cholesterol esterase, described cholesterol oxidase, described metal salt, described horseradish peroxide Compound enzyme, described developer, described 4-AA and described phosphate buffer are mixed to form mixed liquor；Step（2）, add into described mixed liquor described polyethenoxy ether class surfactant and described oxirane- Epoxy propane copolymer surfactant, it is mixed to form described chemical reagent；Step（3）, described reagent pad is put into described chemical reagent, soak 60 ~ 90min, then done at 45 ~ 50 DEG C Dry 40 ~ 60min, described dry chemical reaction plate is made.
- 10. preparation method according to claim 9, it is characterised in that：Described preparation method is also included described sample Product discharge step of the pad in described dry chemical reaction plate.
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| CN109097436A (en) * | 2018-08-10 | 2018-12-28 | 山东博科生物产业有限公司 | A kind of low density lipoprotein cholesterol detection reagent of the single agents of precise and high efficiency |
| CN109916890A (en) * | 2019-04-12 | 2019-06-21 | 吉林省汇酉生物技术股份有限公司 | A kind of dry chemistry reagent piece and preparation method thereof quantitative determining total cholesterol concentration |
| CN110029145A (en) * | 2018-01-11 | 2019-07-19 | 北京瑞正善达生物工程技术有限公司 | Low density lipoprotein cholesterol measures reagent, preparation method and its application method |
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