CN110320086B - Lipoprotein electrophoresis staining solution and preparation method and application thereof - Google Patents
Lipoprotein electrophoresis staining solution and preparation method and application thereof Download PDFInfo
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- CN110320086B CN110320086B CN201910615219.5A CN201910615219A CN110320086B CN 110320086 B CN110320086 B CN 110320086B CN 201910615219 A CN201910615219 A CN 201910615219A CN 110320086 B CN110320086 B CN 110320086B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N2001/302—Stain compositions
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Abstract
The invention provides a lipoprotein electrophoresis staining solution, a preparation method and application thereof. In the preparation process of the lipoprotein electrophoresis staining solution provided by the invention, the lipid dye can be completely dissolved, so that the loss of the lipid dye is avoided on one hand, and the concentration of the completely dissolved lipid dye can be calculated on the other hand, so that the lipoprotein electrophoresis staining solution can be accurately quantified in use. The lipoprotein electrophoresis staining solution provided by the invention has the advantages of long effective period, good staining effect, more obvious staining effect on lipoprotein, higher resolution and more accurate qualitative and quantitative analysis on lipoprotein subtypes.
Description
Technical Field
The invention belongs to the field of biomedical detection, and particularly relates to a lipoprotein electrophoresis staining solution, and a preparation method and application thereof.
Background
When lipoprotein electrophoresis analysis is carried out, a lipid dye is selected for dyeing analysis, because the water solubility of the lipid dye is extremely poor, the preparation process of the commonly used lipoprotein electrophoresis dyeing solution is complicated and takes long time, which often needs several hours or even dozens of hours of preparation time, and the dye is difficult to completely dissolve during the preparation of the dyeing solution, and the excess dye sediment is often removed by centrifugation and/or filtration, so that the final concentration of the dye can not be determined, and the dye dosage is difficult to accurately quantify during the use; in addition, the traditional lipoprotein electrophoresis staining solution has nonuniform solute dispersion, poor stability, easy precipitation and short shelf life, is difficult to maintain the staining effect for a long time, can be stored for only a few days to tens of days, has limitation on the storage temperature, and can further reduce the solubility of lipid dye by utilizing low-temperature storage, so the low-temperature storage is difficult, and the storage is mostly carried out at room temperature; in addition, the conventional lipoprotein electrophoresis staining solution takes a long time to stain lipoproteins, which often takes tens of minutes to several hours, and meanwhile, the lipid dye is difficult to be mixed with water, so that the lipid dye is separated out from the solution during pre-staining (such as plasma pre-staining), which not only reduces the staining effect of lipoproteins, but also increases the workload because the sediment is removed by centrifugation before loading. In a word, the traditional lipoprotein electrophoresis staining solution and the preparation method thereof not only cause the waste of a large amount of manpower and material resources, but also easily cause serious errors in lipoprotein staining analysis.
CN102181173A discloses a Sudan black B staining solution, a preparation method and a use thereof, wherein the Sudan black B staining solution contains ethylene glycol or glycerol and isopropanol, and the volume percentage of the isopropanol contained in the Sudan black B staining solution is more than or equal to 60% based on the total volume of the ethylene glycol or the glycerol and the isopropanol.
Therefore, there is a need to develop a new lipoprotein electrophoretic staining solution with simple preparation method, convenient use and long effective period.
Disclosure of Invention
The invention aims to provide a lipoprotein electrophoresis staining solution and a preparation method and application thereof, and the lipoprotein electrophoresis staining solution provided by the invention has good staining effect on lipoprotein, high resolution and more accurate qualitative and quantitative analysis of lipoprotein subtype; the lipoprotein electrophoresis staining solution provided by the invention is simple to prepare, good in stability and dispersibility, the lipid dye cannot be separated out, the long-term storage can be realized, and the stable staining effect can be maintained for more than 18 months; meanwhile, the storage range is wide, and the product can be stored within the range of 2-60 ℃.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a lipoprotein electrophoresis staining solution, which comprises a lipid dye, an organic solvent, a cosolvent and water.
Wherein the cosolvent is selected from any one of polyethylene glycol, polypropylene glycol or polyvinylpyrrolidone or the combination of at least two of the polyethylene glycol, the polypropylene glycol and the polyvinylpyrrolidone.
The water is double distilled water, so that the dyeing liquid is prevented from being too viscous, the use convenience is improved, and unnecessary loss caused by the viscous adhesion of the dye in the use process can be reduced.
In the invention, a specific cosolvent is added into the lipoprotein electrophoresis dyeing solution, so that the solubility and the stability of the lipid dye in an organic solvent can be improved. On one hand, the solubility of the lipid dye in the organic solvent is increased, so that the lipid dye can be completely dissolved in the organic solvent, the quantitative preparation of the dye can be realized, the unnecessary loss of the lipid dye can be avoided, and the phenomenon that the lipid dye is separated out in the lipoprotein dyeing process can be avoided; on the other hand, the stability of the lipid dye in the solvent is increased, the stable dyeing effect of the lipoprotein electrophoresis dyeing liquid can be maintained, the long-term storage is facilitated, and the temperature range for storage is wide.
In the present invention, the finally obtained lipoprotein electrophoretic staining solution of the present invention has excellent effects only by adding any one or a combination of at least two of polyethylene glycol, polypropylene glycol or polyvinylpyrrolidone, and the addition of other cosolvents does not have any beneficial effects on the stable preservation and testing effects of the lipoprotein electrophoretic staining solution of the present invention.
Preferably, in the lipoprotein electrophoresis staining solution, the concentration of the lipid dye is 0.001-2g/100mL, such as 0.005g/100mL, 0.01g/100mL, 0.05g/100mL, 0.1g/100mL, 0.5g/100mL, 1g/100mL, 1.5g/100mL, etc., more preferably 0.001-1g/100mL, still more preferably 0.001-0.5g/100mL, still more preferably 0.003-0.2g/100 mL.
In the present invention, the unit "g/100 mL" means the mass of the lipid dye contained in 100mL of the lipoprotein electrophoretic staining solution.
Preferably, the volume percentage of the organic solvent is 0.1-30%, for example, 1%, 5%, 10%, 15%, 20%, 25%, etc., more preferably 0.5-20%, still more preferably 0.5-10%, and still more preferably 0.5-5%, based on 100% of the volume of the lipoprotein electrophoretic staining solution.
Preferably, the content of the cosolvent in percentage by mass is 0.15 to 5%, for example, 0.2%, 0.4%, 0.8%, 1.0%, 1.5%, 1.8%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, etc., more preferably 0.25 to 4%, still more preferably 0.5 to 3%, still more preferably 0.5 to 2%, based on 100% by volume of the lipoprotein electrophoresis staining solution.
Preferably, the volume percentage of the water is 0.1-99.8% based on 100% of the volume of the lipoprotein electrophoretic staining solution, such as 1%, 10%, 20%, 40%, 60%, 80%, 90%, etc., more preferably 1-80%, still more preferably 5-60%, and still more preferably 5-40%.
In the present invention, the organic solvent and the cosolvent do not interfere with the electrophoresis of the lipoprotein within the dosage range of the present invention, and do not change the characteristics of the lipoprotein.
Specifically, the cosolvent is selected from any one of or a combination of at least two of PEG200, PEG400, PEG600, PEG800, PEG 1000, PEG 1450, PEG 1500, PEG 2000, PEG4000, PEG6000, PEG8000, PPG200, PPG400, PPG 600, PVP K12, PVPK15, PVP K17, PVP K25, PVP K30, PVP K60 and PVP K90.
Preferably, the cosolvent is selected from any one of or a combination of at least two of PEG400, PEG600, PEG800, PEG 1000, PEG 1450, PEG 1500, PEG 2000, PEG4000, PPG400, PPG 600, PVP K12, PVP K15, PVP K17, PVP K25, PVP K30 and PVPK 60.
Preferably, the cosolvent is selected from any one of or a combination of at least two of PEG 1000, PEG 1450, PEG 1500, PEG 2000, PPG400, PVPK12, PVP K15, PVP K17, PVP K25 and PVP K30.
Preferably, the organic solvent includes any one or a combination of at least two of methanol, ethanol, propanol, isopropanol, acetone, diethyl ether, acetic acid, chloroform, benzene, toluene, phenol, dimethyl sulfoxide, N-dimethylformamide or 1, 4-dioxane, further preferably any one or a combination of at least two of acetone, diethyl ether, acetic acid, phenol, dimethyl sulfoxide, N-dimethylformamide or 1, 4-dioxane, further preferably any one or a combination of at least two of dimethyl sulfoxide, N-dimethylformamide or 1, 4-dioxane.
Preferably, the lipid dye comprises one of sudan i, sudan ii, sudan iii, sudan iv, sudan red 7B, sudan black B, oil red O, wolaisequo blue B, nile blue a, nile red, cell membrane orange red fluorescent probe or sudan red G, further preferably one of sudan i, sudan ii, sudan iii, sudan iv, sudan red 7B, sudan black B, oil red O, wolaisequo blue B, nile blue a or sudan red G, further preferably one of sudan i, sudan ii, sudan iii, sudan iv, sudan red 7B, sudan black B, oil red O or sudan red G, further preferably one of sudan i, sudan iii, sudan iv, sudan red 7B, sudan black B or oil red O, most preferably one of sudan i, sudan iv, sudan red 7B, sudan black B or sudan red O.
In the invention, the lipoprotein electrophoresis staining solution also comprises a densifier.
The densifier is an additive capable of increasing the density of a lipid dye, is a common additive for protein electrophoresis in the prior art, and is used for increasing the density of the lipid dye during prestained dyeing, so that the sample can be conveniently loaded after the prestained lipoprotein is loaded, and the sample can be prevented from floating during loading.
Preferably, the densifier comprises any one or a combination of at least two of ethylene glycol, propylene glycol, glycerol or sucrose, further preferably any one or a combination of at least two of ethylene glycol, propylene glycol or glycerol.
Preferably, the volume percentage of the densifier is 0.01 to 99.7%, for example, 1%, 10%, 20%, 40%, 60%, 80%, 90%, etc., more preferably 10 to 99.5%, still more preferably 10 to 80%, still more preferably 10 to 70%, based on 100% of the volume of the lipoprotein electrophoresis staining solution.
Preferably, when the lipoprotein electrophoresis staining solution is used for staining after lipoprotein electrophoresis, a densifier is not required to be added in the preparation of the staining solution.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
In a second aspect, the present invention provides a method for preparing a lipoprotein electrophoresis staining solution according to the first aspect, the method comprising the steps of:
and mixing the lipid dye, the organic solvent, the cosolvent, the densifier and water to obtain the lipoprotein electrophoresis dyeing liquid.
The preparation method provided by the invention is simple and convenient, consumes short time, and can complete the preparation of the lipoprotein electrophoresis staining solution within 5 min.
As a preferred technical scheme, the method comprises the following steps:
(1) mixing a lipid dye with an organic solvent;
(2) mixing the solution obtained in the step (1) with a cosolvent;
(3) mixing the solution obtained in the step (2) with a densifier;
(4) mixing the solution obtained in the step (3) with water to obtain the lipoprotein electrophoresis staining solution;
wherein the mixing in steps (1) - (4) is each independently selected from 15-30s, e.g., 20s, 25s, etc.; the preparation method is simple and can ensure that the preparation process is finished in 5 min.
The lipoprotein electrophoresis staining solution provided by the invention contains water, so that in order to avoid incomplete dye dissolution, the preparation method provided by the invention needs to add water into a mixing system at last, so that the finally obtained liquid with uniform mixing state can be ensured, and the stable storage can be realized.
In a third aspect, the invention provides the use of the lipoprotein electrophoretic staining solution according to the first aspect in the electrophoretic separation of lipoproteins.
The lipoprotein electrophoresis staining solution provided by the invention can be applied to pre-staining before lipoprotein electrophoresis and staining after the completion of lipoprotein electrophoresis.
Compared with the prior art, the invention has the following beneficial effects:
(1) the cosolvent is added into the lipoprotein electrophoresis dyeing solution, so that the lipid dye can be completely dissolved in the organic solvent, the concentration of the lipid dye can be controlled, and the lipid dye can be accurately quantified when in use, so that the lipoprotein dyeing analysis is more accurate, and the quantitative analysis of lipoprotein subtypes can be realized; meanwhile, the lipid dye can be completely dissolved, so that unnecessary steps such as filtration/centrifugation and the like are reduced, and the loss of the lipid dye is avoided;
(2) the cosolvent increases the solubility and stability of the lipid dye, and the solubility of the lipid dye is increased, so that the lipid dye has better dyeing effect, more obvious dyeing on lipoprotein and higher resolution; meanwhile, the phenomenon of precipitation of lipid dye when dyeing protein can be avoided; on the other hand, the storage time of the staining solution can be prolonged by increasing the stability of the lipid dye, and the stable staining effect of the lipoprotein electrophoresis staining solution obtained by the invention can be maintained for more than 18 months and can be stably stored at the temperature of 2-60 ℃;
(3) the lipoprotein electrophoresis staining solution provided by the invention has short staining time, and can complete the staining of lipoprotein under the conditions of room temperature of 15-20min and 37 ℃ of 5-10 min;
(4) the preparation method provided by the invention is simple and short in time consumption, and the whole preparation process can be completed within 5 min.
Drawings
FIG. 1 is a schematic diagram showing the positions of various types of lipoproteins in a gel column obtained by electrophoretic separation after prestained plasma lipoproteins in example 1 of the present invention.
FIG. 2 is a graph comparing the results of gel electrophoresis after prestained plasma lipoproteins by the lipoprotein electrophoresis staining solutions provided in examples 1, 6 to 7 of the present invention and comparative examples 1,4, 6 and 8, wherein:
FIG. 2A: the gel electrophoresis result graph of the prepared lipoprotein electrophoresis staining solution provided by the embodiment 1 of the invention after prestained plasma lipoprotein;
FIG. 2B: a gel electrophoresis result graph of the lipoprotein electrophoresis staining solution provided by the embodiment 1 of the invention after standing for 18 months at 2 ℃ and pre-staining the plasma lipoprotein;
FIG. 2C: the gel electrophoresis result graph of the prepared staining solution for plasma lipoprotein prestained provided by the embodiment 6 of the invention;
FIG. 2D: a gel electrophoresis result graph of the prepared staining solution for plasma lipoprotein prestained provided by the embodiment 7 of the invention;
FIG. 2E: the gel electrophoresis result graph of the prepared staining solution for plasma lipoprotein prestained provided by the comparative example 1 of the invention;
FIG. 2F: the gel electrophoresis result graph of the prepared staining solution for plasma lipoprotein prestained provided by the comparative example 4 of the invention;
FIG. 2G: the gel electrophoresis result graph of the prepared staining solution for plasma lipoprotein prestained provided by the comparative example 6 of the invention;
FIG. 2H: the gel electrophoresis result graph of the prepared staining solution for plasma lipoprotein prestained provided by the comparative example 8 of the invention;
FIG. 2I: the result of gel electrophoresis of plasma lipoprotein prestained by the staining solution provided by comparative example 8 of the invention after being stored for 7 months at 25 ℃ is shown.
FIG. 3 is a lipoprotein map of gel electrophoresis after staining plasma lipoproteins with the freshly prepared lipoprotein staining solution in example 1 of the present invention.
FIG. 4 is a lipoprotein map of gel electrophoresis after staining plasma lipoproteins with the freshly prepared lipoprotein staining solution of comparative example 8 of the present invention.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The reagents used in the following examples and comparative examples are commercially available from Sigma-Aldrich having a purity of > 99%, unless otherwise specified.
Example 1
A lipoprotein electrophoresis staining solution is prepared by the following steps:
(1) weighing 0.15g of Sudan black B, and placing the Sudan black B in a clean container;
(2) adding 1mL of N, N-dimethylformamide, and vortexing for 30s to completely dissolve Sudan black B;
(3) add 1g PEG 1500 vortex for 30s to mix well;
(4) add 60mL of propylene glycol and 39mL of DDH2And O, vortexing for 1min to fully mix the components to obtain 100mL of lipoprotein electrophoresis staining solution, and storing the lipoprotein electrophoresis staining solution in a brown reagent bottle in a sealed manner.
Example 2
The only difference from example 1 is that in this example, sudan black B was replaced with oil red O.
Example 3
The only difference from example 1 is that in this example, N-dimethylformamide is replaced by 1, 4-dioxane.
Example 4
The only difference from example 1 is that in this example, PEG 1500 was replaced with PVP K30.
Example 5
The only difference from example 1 is that in this example, no densifier, propylene glycol, DDH, is added2The amount of O added was 99 mL.
Example 6
A lipoprotein electrophoresis staining solution is prepared by the following steps:
(1) weighing 0.005g of Sudan black B, and placing in a clean container;
(2) adding 1mL of N, N-dimethylformamide, and vortexing for 30s to completely dissolve Sudan black B;
(3) add 1g PEG 1500 vortex for 30s to mix well;
(4) add 10mL of propylene glycol and 89mL of DDH2And O, vortexing for 1min to fully mix the components to obtain 100mL of lipoprotein electrophoresis staining solution, and storing the lipoprotein electrophoresis staining solution in a brown reagent bottle in a sealed manner.
Example 7
A lipoprotein electrophoresis staining solution is prepared by the following steps:
(1) weighing 2g of Sudan III, and placing in a clean container;
(2) adding 1mL of dimethyl sulfoxide, and vortexing for 30s to completely dissolve Sudan III;
(3) add 1g PVP K12 vortex for 30s and mix well;
(4) adding 60mL of propylene glycol and 39mL of water, swirling for 1min, fully mixing to obtain 100mL of lipoprotein electrophoresis staining solution, and sealing and storing in a brown reagent bottle.
Example 8
A lipoprotein electrophoresis staining solution is prepared by the following steps:
(1) weighing 0.001g of Nile blue A, and placing the Nile blue A in a clean container;
(2) adding 0.1mL of phenol, and vortexing for 30s to completely dissolve the Sudan black B;
(3) add 0.15g PEG4000 vortex for 30s to mix well;
(4) 0.1mL propylene glycol and 99.8mL DDH were added2And O, vortexing for 1min to fully mix the components to obtain 100mL of lipoprotein electrophoresis staining solution, and storing the lipoprotein electrophoresis staining solution in a brown reagent bottle in a sealed manner.
Example 9
A lipoprotein electrophoresis staining solution is prepared by the following steps:
(1) weighing 1g of cell membrane orange-red fluorescent probe, and placing the cell membrane orange-red fluorescent probe in a clean container;
(2) adding 30mL of ethanol, and vortexing for 30s to completely dissolve the Sudan black B;
(3) add 5g PPG 600 vortex for 30s and mix well;
(4) add 69.9mL of propylene glycol and 0.1mL of DDH2And O, vortexing for 1min to fully mix the components to obtain 100mL of lipoprotein electrophoresis staining solution, and storing the lipoprotein electrophoresis staining solution in a brown reagent bottle in a sealed manner.
Example 10
The only difference from example 1 is that 0.5g of PEG 1500, 0.5g of PPG400 and 0.5g of PVP K15 were added in step (3) of this example.
Comparative example 1
The difference from example 1 is that in this example, DDH was not added2The amount of O, N, N-dimethylformamide used was 40 mL.
Comparative example 2
The only difference from example 1 is that in this example, N-dimethylformamide is not added, and at the same time, DDH2The amount of O added was 40 mL.
Comparative example 3
The only difference from example 1 is that, in this example, no PEG 1500 is added.
Comparative example 4
A lipoprotein electrophoresis staining solution is prepared by the following steps:
(1) weighing 0.15g of Sudan black B, and placing the Sudan black B in a clean container;
(2) adding 1mL of N, N-dimethylformamide, and vortexing for 30s to completely dissolve Sudan black B;
(3) adding 0.1mL surfactant Triton X-100, and vortexing for 30s to mix thoroughly;
(4) 60mL of propylene glycol and 38.9mL of DDH were added2And O, vortexing for 1min to fully mix the components to obtain 100mL of lipoprotein electrophoresis staining solution, and storing the lipoprotein electrophoresis staining solution in a brown reagent bottle in a sealed manner.
Comparative example 5
The only difference from comparative example 4 was that in this example Triton X-100 was replaced with Pluronic F-127.
Comparative example 6
The only difference from comparative example 4 is that in this example, Triton X-100 was replaced with Tween 20.
Comparative example 7
The only difference from comparative example 4 is that in this example, Triton X-100 was replaced with Sodium Dodecyl Sulfate (SDS).
Comparative example 8
The lipoprotein electrophoresis staining solution is prepared according to the method disclosed in CN102181173A, and the specific method is as follows:
adding 100mL of isopropanol and ethylene glycol mixed solution (isopropanol: ethylene glycol 4:1) into 0.5g of Sudan black B, adding none of the other reagents, mixing for 5min, performing water bath at 37 ℃ for 24h, and centrifuging to remove sediments to obtain the lipoprotein electrophoresis staining solution.
Comparative example 9
The present comparative example provides a preparation method commonly used at present to prepare a lipoprotein electrophoretic staining solution, and is different from comparative example 8 only in that a mixed solution of isopropyl alcohol and ethylene glycol is replaced by propylene glycol.
Performance testing
The lipoprotein electrophoresis staining solutions provided in examples 1-10 and comparative examples 1-9 were subjected to performance testing as follows:
(1) stability: standing the sample at 2 ℃, 25 ℃, 37 ℃ and 60 ℃ for 18 months, then observing whether the lipid dye is separated out of the solution, and if the observation phenomenon is not obvious, centrifuging the staining solution at 3000r/min for 1min, and then observing whether the dye is precipitated at the tube bottom of the centrifugal tube by naked eyes;
(2) whether dye is separated out in the dyeing process: after lipoprotein dyeing is finished, centrifuging the dyeing mixed solution at 3000r/min for 1min, and observing whether dye precipitates at the tube bottom of a centrifugal tube by naked eyes;
(3) and (3) testing dyeing effect: mixing the staining solution with plasma, shaking and mixing uniformly, pre-staining plasma lipoprotein in a water bath at 37 ℃ for 5min, loading the pre-stained mixed solution completely, and performing electrophoretic separation, scanning and qualitative and quantitative analysis on the plasma lipoprotein by referring to the method described in CN 109374717A.
Wherein, in the same amount of plasma, in order to ensure the same total amount of the lipid dye used, 5 μ L of the staining solutions provided in examples 1-5 and 10 and comparative examples 1-7 and 25 μ L of plasma were mixed, 150 μ L of the staining solution provided in example 6 and 25 μ L of plasma were mixed, 0.375 μ L of the staining solution provided in example 7 and 25 μ L of plasma were mixed, 750 μ L of the staining solution provided in example 8 and 25 μ L of plasma were mixed, and 0.75 μ L of the staining solution provided in example 9 and 25 μ L of plasma were mixed, and since the final concentration of the lipid dye could not be determined in comparative examples 8-9, the conventional staining method in CN102181173A and 2.5 μ L of the staining solutions provided in comparative examples 8-9 and 25 μ L of plasma were mixed.
The plasma used was from Shanghai Bao Teng medical laboratory Co., Ltd, blood collected from volunteers after 12h fasting, and plasma separated within 2 h.
FIG. 1 is a schematic diagram showing the positions of various types of lipoproteins obtained by electrophoretic separation in a gel column according to the method described in CN109374717A after plasma lipoproteins are prestained in example 1 of the present invention, wherein the distribution of various plasma lipoprotein subtypes and electrophoretic bands thereof can be clearly observed in FIG. 1.
FIG. 2 is a graph showing the results of gel electrophoresis after prestained plasma lipoproteins by the lipoprotein electrophoresis staining solutions provided in examples 1, 6 to 7 of the present invention and comparative examples 1,4, 6 and 8. Wherein, fig. 2A is a graph of a gel electrophoresis result of a pre-staining plasma lipoprotein with a prepared staining solution for lipoprotein electrophoresis provided in example 1 of the present invention, fig. 2B is a graph of a gel electrophoresis result of a pre-staining plasma lipoprotein with a prepared staining solution provided in example 1 of the present invention after standing at 2 ℃ for 18 months, fig. 2C is a graph of a gel electrophoresis result of a pre-staining plasma lipoprotein with a prepared staining solution provided in example 6 of the present invention, fig. 2D is a graph of a gel electrophoresis result of a pre-staining plasma lipoprotein with a prepared staining solution provided in example 7 of the present invention, fig. 2E is a graph of a gel electrophoresis result of a pre-staining plasma lipoprotein with a prepared staining solution provided in comparative example 1 of the present invention, fig. 2F is a graph of a gel electrophoresis result of a pre-staining plasma lipoprotein with a prepared staining solution provided in comparative example 4 of the present invention, and fig. 2G is a graph of a gel electrophoresis result of a pre-staining plasma lipoprotein with a prepared staining solution provided in comparative example 6 of the present invention, FIG. 2H is a graph showing the results of gel electrophoresis of the pre-staining of plasma lipoproteins with the freshly prepared staining solution provided in comparative example 8 of the present invention, and FIG. 2I is a graph showing the results of gel electrophoresis of the pre-staining of plasma lipoproteins with the staining solution provided in comparative example 8 of the present invention after being stored at 25 ℃ for 7 months.
As can be seen from the comparison between fig. 2A and fig. 2B, the staining effect of the lipoprotein electrophoretic staining solution of the present invention is not changed even when the lipoprotein electrophoretic staining solution is stored at 2 ℃ for 18 months, and as can be seen from fig. 2C, the lipoprotein electrophoretic staining solution provided by the present invention can be applied even when the concentration of the lipid dye is low (0.005g/100mL), and has a good staining effect, and as can be seen from fig. 2A, fig. 2C and fig. 2D, even when the concentration of the prepared dye is different, the same staining effect can be achieved by changing the amount of the staining solution. As can be seen from fig. 2E, 2F and 2G, the staining solutions provided in comparative example 1,4 and 6 have poor staining effects on lipoproteins, and separation of lipoprotein subtypes is difficult to achieve; as is apparent from FIGS. 2H and 2I, the dye of comparative example 8 was very poor in dyeing effect after being stored at 25 ℃ for 7 months.
As shown in FIG. 2, compared with the prior art, the lipoprotein electrophoresis staining solution of the present invention has the advantages of good stability, long maintenance time of staining effect and excellent staining stability. Meanwhile, as can be seen from fig. 2A and 2H, the staining effect of the lipoprotein electrophoresis staining solution provided by the present invention is equivalent to, even better than, the staining effect of the staining solution in the prior art.
FIG. 3 is a lipoprotein map of gel electrophoresis after staining plasma lipoprotein with the freshly prepared lipoprotein electrophoretic staining solution in example 1 of the present invention, FIG. 4 is a lipoprotein map of gel electrophoresis after staining plasma lipoprotein with the freshly prepared lipoprotein electrophoretic staining solution in comparative example 8 of the present invention, and it can be seen from the comparison of FIG. 3 and FIG. 4 that when the lipoprotein electrophoretic staining solution of the present invention is used for lipoprotein analysis, more small sharp peaks of the curve can be clearly seen in the map, and less LDL peaks of comparative example 8, compared with comparative example 8, which shows that the resolution of staining with the lipoprotein electrophoretic staining solution obtained by the present invention is higher, so that qualitative and quantitative analysis of lipoprotein subtype is more accurate.
The results of the tests on examples 1-10 and comparative examples 1-9 are shown in Table 1:
TABLE 1
The embodiment and the performance test show that the lipoprotein electrophoresis staining solution provided by the invention has good stability, is stable after being respectively kept still for 18 months at 2 ℃, 25 ℃, 37 ℃ and 60 ℃, and has no precipitation of lipid dye, and meanwhile, the phenomenon of precipitation of lipid dye is avoided in the staining process, and the staining effect on lipoprotein is still stable after being kept still for 18 months. In the preparation process of the lipoprotein electrophoresis staining solution provided by the invention, the lipid dye can be completely dissolved, so that the loss of the lipid dye is avoided on one hand, and the concentration of the completely dissolved lipid dye can be calculated on the other hand, so that the lipoprotein electrophoresis staining solution can be accurately quantified in use. The lipoprotein electrophoresis staining solution provided by the invention has good staining effect, more obvious staining effect on lipoprotein, higher resolution and more accurate qualitative and quantitative analysis of lipoprotein subtype.
In comparative examples 1,4 and 6, although the dye liquor of comparative example 1 has a long shelf life and remains stable after standing at 2 ℃, 25 ℃, 37 ℃ and 60 ℃ for 18 months, due to the excessive content of the organic solvent in the dye liquor of comparative example 1, the addition of the polyoxyethylene-type nonionic surfactant in the dye liquor of comparative example 4 and the dye liquor of comparative example 6 causes the change of the lipoprotein characteristics or the dissolution of the lipoprotein, so the dyeing effect of the lipoprotein is poor, and the separation of lipoprotein subtypes is difficult to realize, namely the lipoprotein electrophoresis dye liquor provided by comparative examples 1,4 and 6 interferes with the electrophoresis of the lipoprotein to generate deviation. As can be seen from comparative example 7, the anionic surfactant Sodium Dodecyl Sulfate (SDS) commonly used in protein electrophoresis does not exert a beneficial effect on the stable preservation of the staining solution for lipoprotein electrophoresis of the present invention. As can be seen from the comparison of example 1 and comparative examples 1-3, the organic solvent, cosolvent and water of the present invention are all one but not indispensable; as can be seen from the comparison of example 1 and comparative examples 8 to 9, the present invention has better stability and more obvious staining of lipoproteins and higher resolution than the prior art; as can be seen from the comparison of example 1 and comparative examples 4 to 7, the selection of the co-solvent is specific in the present invention.
The applicant states that the present invention is illustrated by the above examples of the lipoprotein electrophoretic staining solution of the present invention and the preparation method and application thereof, but the present invention is not limited to the above process steps, i.e. it does not mean that the present invention must rely on the above process steps to be carried out. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (7)
1. A lipoprotein electrophoresis staining fluid comprises a lipid dye and an organic solvent, and is characterized by also comprising a cosolvent and water;
wherein the cosolvent is selected from any one of polyethylene glycol, polypropylene glycol or polyvinylpyrrolidone or the combination of at least two of the polyethylene glycol, the polypropylene glycol and the polyvinylpyrrolidone.
2. The lipoprotein electrophoresis staining solution of claim 1, wherein the concentration of the lipid dye in the lipoprotein electrophoresis staining solution is 0.001-2g/100 mL;
the volume percentage of the organic solvent is 0.1-30% calculated by the volume of the lipoprotein electrophoresis staining solution as 100%;
the mass percentage of the cosolvent is 0.15-5% by taking the volume of the lipoprotein electrophoresis staining solution as 100%;
the volume percentage of the water is 0.1-99.8% based on 100% of the volume of the lipoprotein electrophoresis staining solution.
3. The lipoprotein electrophoretic staining solution according to claim 1, wherein the organic solvent comprises any one or a combination of at least two of methanol, ethanol, propanol, isopropanol, acetone, diethyl ether, acetic acid, chloroform, benzene, toluene, phenol, dimethyl sulfoxide, N-dimethylformamide, or 1, 4-dioxane;
the lipid dye comprises one of Sudan I, Sudan II, Sudan III, Sudan IV, Sudan red 7B, Sudan black B, oil red O, Wolaxideblue B, Nile blue A, Nile red, cell membrane orange red fluorescent probe or Sudan red G.
4. The lipoprotein electrophoresis staining solution of claim 1, further comprising a densifier;
the densifier comprises any one or the combination of at least two of ethylene glycol, propylene glycol, glycerol or sucrose;
the volume percentage of the densifier is 0.01-99.7% based on 100% of the volume of the lipoprotein electrophoresis staining solution.
5. The method for preparing the lipoprotein electrophoretic staining solution according to any one of claims 1 to 4, wherein the method comprises the following steps:
and mixing the lipid dye, the organic solvent, the cosolvent, the densifier and water to obtain the lipoprotein electrophoresis dyeing liquid.
6. The method of claim 5, comprising the steps of:
(1) mixing a lipid dye with an organic solvent;
(2) mixing the solution obtained in the step (1) with a cosolvent;
(3) mixing the solution obtained in the step (2) with a densifier;
(4) mixing the solution obtained in the step (3) with water to obtain the lipoprotein electrophoresis staining solution;
wherein the mixing in steps (1) - (4) is independently selected from 15-30 s.
7. Use of a lipoprotein electrophoretic staining solution according to any one of claims 1-4 for electrophoretic separation of lipoproteins.
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