CN113281520A - Preparation method of reagent ball for determining total serum protein, reagent ball and microfluidic chip - Google Patents
Preparation method of reagent ball for determining total serum protein, reagent ball and microfluidic chip Download PDFInfo
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- 102000004506 Blood Proteins Human genes 0.000 title claims abstract description 47
- 108010017384 Blood Proteins Proteins 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title abstract description 14
- 239000000546 pharmaceutical excipient Substances 0.000 claims abstract description 33
- 239000007853 buffer solution Substances 0.000 claims abstract description 31
- LJCNRYVRMXRIQR-OLXYHTOASA-L potassium sodium L-tartrate Chemical compound [Na+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O LJCNRYVRMXRIQR-OLXYHTOASA-L 0.000 claims abstract description 30
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- 238000004108 freeze drying Methods 0.000 claims abstract description 15
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- 238000000034 method Methods 0.000 claims description 20
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- 238000006243 chemical reaction Methods 0.000 claims description 15
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- BHQCQFFYRZLCQQ-UHFFFAOYSA-N (3alpha,5alpha,7alpha,12alpha)-3,7,12-trihydroxy-cholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)C(O)C2 BHQCQFFYRZLCQQ-UHFFFAOYSA-N 0.000 claims description 13
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
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- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 9
- 238000002156 mixing Methods 0.000 claims description 9
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- 229920001223 polyethylene glycol Polymers 0.000 claims description 9
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 claims description 7
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- CMCBDXRRFKYBDG-UHFFFAOYSA-N 1-dodecoxydodecane Chemical compound CCCCCCCCCCCCOCCCCCCCCCCCC CMCBDXRRFKYBDG-UHFFFAOYSA-N 0.000 claims description 4
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- 238000001514 detection method Methods 0.000 abstract description 33
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- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 description 9
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- 230000000694 effects Effects 0.000 description 6
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- 238000002835 absorbance Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 3
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- UWJDLABDOBGBFS-UHFFFAOYSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;sodium Chemical compound [Na].[Na].OC(=O)CC(O)(C(O)=O)CC(O)=O UWJDLABDOBGBFS-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 2
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- CIWBSHSKHKDKBQ-UHFFFAOYSA-N 2-(1,2-dihydroxyethyl)-3,4-dihydroxy-2h-furan-5-one Chemical compound OCC(O)C1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-UHFFFAOYSA-N 0.000 description 1
- DYQXMFHEZICODL-UHFFFAOYSA-N 2-[2-[2-(2-hydroxyethoxy)ethoxy]ethoxy]ethyl dodecanoate Chemical compound CCCCCCCCCCCC(=O)OCCOCCOCCOCCO DYQXMFHEZICODL-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/683—Total protein determination, e.g. albumin in urine involving metal ions
- G01N33/6833—Copper, e.g. Folin-, Lowry-, biuret methods
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Bioinformatics & Computational Biology (AREA)
- Microbiology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The embodiment of the invention relates to the technical field of medical detection, in particular to a preparation method of a reagent ball for measuring total serum protein, the reagent ball and a microfluidic chip. Wherein the mixed solution comprises the following components: 40-200mmol/L buffer solution, 10-40g/L blue vitriol, 20-80g/L potassium sodium tartrate, 10-40g/L iodide, 5-20g/L non-ionic surfactant, 0.1-10g/L stabilizer and 10-100g/L excipient. The excipient in the dosage range can ensure the shape and the re-melting solubility of the reagent ball, and is beneficial to the complete freeze-drying of the reagent ball, so that the reagent ball has higher stability and precision.
Description
Technical Field
The embodiment of the invention relates to the technical field of medical detection, in particular to a preparation method of a reagent ball for determining total serum protein, the reagent ball and a microfluidic chip.
Background
Total Protein (TP) of serum comprises albumin and globulin, has important physiological functions in the body, can maintain normal colloid osmotic pressure and PH of blood, transports various metabolites, and has multiple functions of immunization, nutrition and the like. The total serum protein can reflect the synthetic function of the liver, and when the liver is diseased, the total serum protein is obviously increased. Therefore, the determination of total serum protein is a major test item for liver diseases.
At present, the biuret method is the most commonly used method for measuring total serum protein in clinical laboratories, however, the sensitivity of the biuret method is poor, and the biuret method needs to depend on a large-scale full-automatic biochemical analyzer, and is difficult to be applied to patients for timely diagnosis (Point of care, POCT). Although the freeze-dried reagent ball is used for detecting other items, the conventional freeze-dried reagent ball has poor morphology and is difficult to completely freeze-dry, so that the conventional freeze-dried reagent ball has low stability and accuracy.
Disclosure of Invention
The technical problem mainly solved by the embodiment of the invention is to provide a preparation method of a reagent ball for determining total serum protein, the reagent ball and a microfluidic chip.
In order to solve the above technical problems, in a first aspect, an embodiment of the present invention provides a method for preparing a reagent ball for measuring total serum protein, including:
mixing buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, iodide, nonionic surfactant, stabilizer, excipient and water to form mixed solution;
dropping the liquid drops of the mixed liquid into liquid nitrogen to enable the liquid drops to form ice balls;
freeze-drying the ice ball to prepare the reagent ball for measuring the total serum protein;
wherein the mixed solution comprises the following components: 40-200mmol/L buffer solution, 10-40g/L copper sulfate pentahydrate, 20-80g/L potassium sodium tartrate, 10-40g/L iodide, 5-20g/L nonionic surfactant, 0.1-10g/L stabilizer and 10-100g/L excipient, wherein the stabilizer comprises sodium carbonate and cholic acid.
In some embodiments, the mixing of the buffer, copper sulfate pentahydrate, sodium potassium tartrate, iodide, nonionic surfactant, stabilizer, excipient, and water to form a mixture comprises:
adding the buffer to a first predetermined amount of water;
after the buffer solution is completely dissolved in the water, sequentially adding copper sulfate pentahydrate, sodium potassium tartrate and iodide to form a first solution;
sequentially adding a nonionic surfactant, a stabilizer and an excipient into the first solution, and adding a second preset amount of water into the first solution to form a third preset amount of the mixed solution.
In some embodiments, the excipient comprises at least one of mannitol, inositol, trehalose, PEG3350, PEG8000, polyvinylpyrrolidone, dextran 1 ten thousand, dextran 4 ten thousand.
In some embodiments, the buffer comprises at least one of a sodium hydroxide buffer, a potassium hydroxide buffer, a disodium citrate buffer.
In some embodiments, the nonionic surfactant comprises at least one of polyethylene glycol octyl phenyl ether, tetrapolyethylene glycol monolauryl ether.
In some embodiments, the ice ball has a volume of 2.5ul to 3.5 ul.
In order to solve the above technical problems, in a second aspect, the present invention provides a reagent ball prepared by the method for preparing a reagent ball for measuring total serum protein according to the first aspect.
In order to solve the above technical problem, in a third aspect, the present invention provides a microfluidic chip, including a chip body and the reagent ball as described in the second aspect, wherein the reagent ball is disposed inside the chip body.
In order to solve the above technical problem, in a fourth aspect, an embodiment of the present invention provides a biochemical analyzer, including an analyzer body, a reaction tank, and the microfluidic chip according to the third aspect, wherein the reaction tank is disposed in the analyzer body, and the microfluidic chip is mounted in the reaction tank.
The embodiment of the invention has the following beneficial effects: different from the prior art, the preparation method of the reagent ball for measuring total serum protein provided by the embodiment of the invention is that a certain amount of buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, iodide, nonionic surfactant, stabilizer, excipient and water are mixed to form a mixed solution, then the mixed solution is dropped into liquid nitrogen in the form of liquid drops, so that the liquid drops form ice balls, and then the ice balls are frozen and dried to prepare the spherical reagent ball for measuring total serum protein. Wherein the mixed solution comprises the following components: 40-200mmol/L buffer solution, 10-40g/L copper sulfate pentahydrate, 20-80g/L potassium sodium tartrate, 10-40g/L iodide, 5-20g/L nonionic surfactant, 0.1-10g/L stabilizer and 10-100g/L excipient, wherein the stabilizer comprises sodium carbonate and cholic acid. The sodium potassium tartrate provides an alkaline environment, so that copper ions in the blue-violet sulfate and peptide bonds of total proteins are subjected to a complex reaction to form a blue-violet complex, the color intensity of the blue-violet complex is combined to evaluate the content of the total proteins, the iodide is helpful for preventing the complex from being automatically reduced, the nonionic surfactant can increase the contact and combination of the total proteins and the copper ions of serum, and simultaneously can reduce the turbidity of the serum to a certain extent, so that the anti-interference effect is achieved, and the detection accuracy is improved; on the other hand, sodium carbonate and cholic acid in the stabilizer form cholate, and the cholate can well play roles in emulsifying, thickening and maintaining the activity of each component; in addition, the excipient in the dosage range can ensure the shape and the re-melting solubility of the reagent ball, and is beneficial to completely freeze-drying the reagent ball. Namely, the reagent ball for measuring the total serum protein prepared by the method has better shape and meltdown solubility, and can be completely frozen and dried, thereby having higher stability and precision.
Drawings
One or more embodiments are illustrated by way of example in the accompanying drawings, which correspond to the figures in which like reference numerals refer to similar elements and which are not to scale unless otherwise specified.
FIG. 1 is a schematic flow chart of a method for preparing reagent spheres for determination of total serum protein according to an embodiment of the present invention;
FIG. 2 is a schematic view of a sub-process of step S10 shown in FIG. 1;
FIG. 3 is a schematic line of fit of correlation equations in clinical correlation analysis;
FIG. 4 is a line schematic of the fit of the correlation equations in the linear range test.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
It should be noted that, if not conflicted, the various features of the embodiments of the invention may be combined with each other within the scope of protection of the present application. Additionally, while functional block divisions are performed in apparatus schematics, with logical sequences shown in flowcharts, in some cases, steps shown or described may be performed in sequences other than block divisions in apparatus or flowcharts. Further, the terms "first," "second," "third," and the like, as used herein, do not limit the data and the execution order, but merely distinguish the same items or similar items having substantially the same functions and actions.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The embodiment of the present invention provides a method for preparing a reagent ball for measuring total serum protein, which can prepare a spherical reagent for measuring total serum protein, and please refer to fig. 1, wherein the method comprises the following steps:
s10: mixing buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, iodide, nonionic surfactant, stabilizer, excipient and water to form a mixed solution.
In some embodiments, the buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, iodide, nonionic surfactant, stabilizer, excipient, and water (each component) are measured separately, and then the components are mixed well to obtain a mixed solution.
In order to fully mix the mixed solution and accurately contain the components, in some embodiments, referring to fig. 2, the step S10 specifically includes the following steps:
s11: the buffer is added to a first predetermined amount of water.
S12: and after the buffer solution is completely dissolved in the water, sequentially adding copper sulfate pentahydrate, sodium potassium tartrate and iodide to form a first solution.
S13: sequentially adding a nonionic surfactant, a stabilizer and an excipient into the first solution, and adding a second preset amount of water into the first solution to form a third preset amount of the mixed solution.
Specifically, 800ml of water is added into a 1L beaker, namely the first preset amount is 800ml, buffer solution components are weighed and added into the beaker filled with 800ml of water, and copper sulfate pentahydrate, potassium sodium tartrate and iodide are sequentially added after the buffer solution is completely dissolved in the water to form a first solution. Thus, the water, the buffer solution, the copper sulfate pentahydrate, the sodium potassium tartrate and the iodide can be completely and fully mixed.
Then, adding a nonionic surfactant, a stabilizer and an excipient into the first solution to fully mix the nonionic surfactant, the stabilizer and the excipient with the first solution, and finally, adding a second preset amount of water to fix the volume of the solution in the beaker to 1L, so as to obtain 1L (third preset amount) of mixed solution.
Based on the fact that the mixed liquid is composed of a plurality of components, if the amount of water is determined once, the subsequent total amount (namely, the third preset amount) is easy to exceed the standard, and the content of each component is inaccurate. In this example, it is beneficial to formulate an accurate mixture of the components by adding a predetermined amount of water twice. The components are added in batches, so that the components can be uniformly mixed, and the detection precision is improved.
In addition, the mixed solution comprises the following components: 40-200mmol/L of buffer solution, 10-40g/L of copper sulfate pentahydrate, 20-80g/L of sodium potassium tartrate, 10-40g/L of iodide, 5-20g/L of nonionic surfactant, 0.1-10g/L of stabilizer and 10-100g/L of excipient, wherein the stabilizer comprises sodium carbonate and cholic acid.
Wherein the buffer solution comprises at least one of sodium hydroxide buffer solution, potassium hydroxide buffer solution and citric acid disodium buffer solution. It will be appreciated that the buffer provides an alkaline environment for the reagent to allow the other components to function effectively.
Wherein, the copper ions in the blue-violet copper sulfate can generate complex reaction with peptide bonds in the serum total protein under the alkaline environment to form a blue-violet complex. The blue-violet complex is irradiated by spectrophotometry, a special absorption peak exists at 540nm, and the color intensity of the blue-violet complex is in direct proportion to the concentration of total protein in a sample in a certain range, so that the content of the total protein in serum can be calculated from the measured absorbance value.
Under the alkaline environment, the potassium sodium tartrate participates in the formation of a bluish purple compound with copper ions and peptide bonds, maintains the solubility of the bluish purple compound, and ensures that the copper ions and the peptide bonds fully react.
Wherein the nonionic surfactant comprises at least one of polyethylene glycol octyl phenyl ether and tetrapolyethylene glycol monolauryl ether. It can be understood that the nonionic surfactant can increase the contact and combination between the macromolecular protein and the anhydrous copper sulfate and the potassium sodium tartrate, and can reduce the lipid turbidity of the serum to a certain extent at the same time, thereby playing a role in resisting interference.
Wherein the iodide may be potassium iodide, which helps to prevent auto-reduction of alkaline copper complex (blue-violet complex) and maintain divalent Cu2+Concentration, ensuring a wide linear range.
Wherein, sodium carbonate in the stabilizer and cholic acid form cholate, and the cholate can well play roles in emulsifying, thickening and maintaining the activity of each component.
In addition, the excipient comprises at least one of mannitol, inositol, trehalose, PEG3350, PEG8000, polyvinylpyrrolidone, dextran 1 ten thousand, and dextran 4 ten thousand. It is understood that the excipient can give the reagent ball a good appearance, and the reagent ball is loosened, so that the reagent ball has good solubility and sufficiently reacts with the sample after contacting the test sample. For example, the excipient comprises PEG3350, or comprises PEG8000, or comprises polyvinylpyrrolidone.
S20: and dropping the liquid drops of the mixed liquid into liquid nitrogen to enable the liquid drops to form ice balls.
After the mixed liquid is prepared, the liquid drops of the mixed liquid can be dropped in liquid nitrogen through a dispenser, so that the liquid drops are coagulated into ice balls in the liquid nitrogen. The size of the liquid drop of the mixed liquid dropped into the liquid nitrogen can be adjusted by those skilled in the art according to actual needs, and the volume of the ice ball can be adjusted by controlling the size of the liquid drop. Optionally, in some embodiments, the ice ball has a volume of 2.5ul to 3.5 ul.
S30: and (3) freeze-drying the ice ball to prepare the reagent ball for measuring the total serum protein.
And after obtaining the ice ball, placing the ice ball in a vacuum freeze dryer for freeze drying to obtain a reagent ball for measuring the total serum protein, and collecting and storing the reagent ball for measuring the total serum protein in a dry aluminum bottle after nitrogen repression.
Wherein, freeze drying means that the ice ball is cooled and frozen into solid in advance, and the sublimation performance of water is utilized under the condition of low temperature and reduced pressure to dehydrate the ice ball at low temperature so as to achieve the drying purpose. After freeze drying, all components (buffer solution, nonionic detergent, anionic dye and excipient) except water in the ice ball are left in an ice shelf during freezing, so that the ice ball after freeze drying is loose and porous and has unchanged volume, and the ice ball is always in a frozen state before drying, and meanwhile, ice crystals are uniformly distributed in a substance, so that the concentration phenomenon caused by dehydration in the sublimation process can be avoided. The reagent ball obtained after freeze drying is spongy, loose and porous, the volume of the reagent ball is basically unchanged with that of the ice ball before drying, and the reagent ball is easy to dissolve in water and restore to the original shape.
In this example, by providing the excipient and the stabilizer in a high content, the form and the reconstitution solubility of the reagent beads can be ensured, which is advantageous for complete lyophilization of the reagent beads. Therefore, the reagent ball for measuring the total serum protein prepared by the method has better shape and meltdown solubility, and can be completely lyophilized, thereby having higher stability and precision and realizing timely diagnosis.
The detection principle of the reagent ball prepared by the preparation method of the reagent ball for determining the total serum protein provided by the embodiment of the invention is as follows: the detection sample is contacted with the albumin determination reagent ball, so that the albumin determination reagent ball is dissolved and fully reacts with the detection sample, copper ions in blue-violet sulfate can generate a complex reaction with peptide bonds in serum total protein under the alkaline environment provided by a buffer solution to form a blue-violet complex, the full reaction and stability of the blue-violet complex can be maintained under the action of sodium potassium tartrate, iodide and a nonionic surfactant, and the reagent ball has good shape and complex solubility under the action of an excipient and a stabilizer. The blue-violet complex is irradiated by spectrophotometry, a special absorption peak exists at 540nm, and the color intensity of the blue-violet complex is in direct proportion to the concentration of total protein in a sample in a certain range, so that the content of the total protein in serum can be calculated from the measured absorbance value.
The embodiment of the invention also provides a reagent ball, which is prepared by the preparation method of the reagent ball for determining total serum protein in the above embodiment, and has the same structure and function as the reagent ball prepared by the preparation method of the reagent ball for determining total serum protein in the above embodiment, and the details are not repeated.
The embodiment of the invention also provides a microfluidic chip which comprises a chip body and the reagent ball provided by any one of the embodiments, wherein the reagent ball is arranged in the chip body. In some embodiments, the chip body is formed by adhering an injection-molded plastic substrate and an optical film through an adhesive layer, and the chip body comprises a sample tank, a dilution liquid tank, a quantification tank, a mixing tank, a waste liquid tank, a liquid flow channel, a colorimetric hole and the like. The chip body also comprises a plurality of colorimetric holes for storing the reagent balls, and the detection sample enters the colorimetric holes and then undergoes chemical reaction with the reagent balls.
The embodiment of the invention also provides a biochemical analyzer, which comprises an analyzer body, a reaction tank and the microfluidic chip, wherein the reaction tank is arranged in the analyzer body, and the microfluidic chip can be arranged in the reaction tank, so that a detection sample in the microfluidic chip can be analyzed and detected. The structure and the function of the microfluidic chip are completely the same as those of the microfluidic chips in the embodiments, and are not described in detail herein.
To further illustrate the technical solution of the present invention, several examples of the preparation method of the reagent ball for measuring total serum protein according to the present invention are provided below.
Example 1:
in this example, the buffer comprises sodium hydroxide buffer, the iodide comprises potassium iodide, the non-ionic surfactant comprises polyethylene glycol octyl phenyl ether, the stabilizer comprises sodium carbonate and cholic acid, and the excipient comprises PEG 8000. The preparation method of the reagent ball for measuring the total serum protein comprises the following steps: mixing a certain amount of sodium hydroxide buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, potassium iodide, polyethylene glycol octyl phenyl ether, sodium carbonate, cholic acid, PEG8000 and water to form a mixed solution, dripping the liquid drops of the mixed solution into liquid nitrogen to form an ice ball with the volume of about 3.0ul, and freeze-drying the ice ball to obtain the reagent ball for measuring the total protein of the serum. And preparing the microfluidic chip by using the reagent ball for determining total serum protein.
Specifically, in this embodiment, the mixed solution includes the following components: 40-200mmol/L of sodium hydroxide buffer solution, 10-40g/L of copper sulfate pentahydrate, 20-80g/L of potassium sodium tartrate, 0.1-10g/L of potassium iodide, 5-20g/L of polyethylene glycol octyl phenyl ether, 0.1-10g/L of sodium carbonate, 0.1-10g/L of cholic acid, PEG 8000: 10-100 g/L.
Example 2:
in this example, the buffer comprises sodium hydroxide buffer, the iodide comprises potassium iodide, the non-ionic surfactant comprises tetrapolyethylene glycol monolaurate, the stabilizer comprises sodium carbonate and cholic acid, and the excipient comprises PEG 3350. The preparation method of the reagent ball for measuring the total serum protein comprises the following steps: mixing a certain amount of sodium hydroxide buffer solution, blue vitriol, potassium sodium tartrate, potassium iodide, tetraethylene glycol monolaurate, sodium carbonate, cholic acid, PEG3350 and water to form a mixed solution, dripping the liquid drops of the mixed solution into liquid nitrogen to form an ice ball with the volume of about 2.5ul, and freeze-drying the ice ball to obtain the reagent ball for measuring the total protein of the serum. And preparing the microfluidic chip by using the reagent ball for determining total serum protein.
Specifically, in this embodiment, the mixed solution includes the following components: 40-200mmol/L of sodium hydroxide buffer solution, 10-40g/L of copper sulfate pentahydrate, 20-80g/L of sodium potassium tartrate, 0.1-10g/L of potassium iodide, 5-20g/L of tetrapolyethylene glycol monolauryl ether, 0.1-10g/L of sodium carbonate, 0.1-10g/L of cholic acid, PEG 3350: 10-100 g/L.
Example 3:
in this example, the buffer comprises disodium citrate buffer, the iodide comprises potassium iodide, the non-ionic surfactant comprises octyl phenyl ether of polyethylene glycol, the stabilizer comprises sodium carbonate and cholic acid, and the excipient comprises polyvinylpyrrolidone. The preparation method of the reagent ball for measuring the total serum protein comprises the following steps: mixing a certain amount of disodium citrate buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, potassium iodide, polyethylene glycol octyl phenyl ether, sodium carbonate, polyvinylpyrrolidone and water to form a mixed solution, dripping the liquid drops of the mixed solution into liquid nitrogen to form an ice ball with the volume of about 3.5ul, and freeze-drying the ice ball to prepare the reagent ball for measuring the total protein of the serum. And preparing the microfluidic chip by using the reagent ball for determining total serum protein.
Specifically, in this embodiment, the mixed solution includes the following components: 40-200mmol/L of citric acid disodium buffer solution, 10-40g/L of copper sulfate pentahydrate, 20-80g/L of potassium sodium tartrate, 0.1-10g/L of potassium iodide, 5-20g/L of polyethylene glycol octyl phenyl ether, 0.1-10g/L of sodium carbonate, 0.1-10g/L of cholic acid and 10-100g/L of polyvinylpyrrolidone.
The performance of the reagent ball prepared in example 1 of the present invention will be described below with reference to specific test and comparative experiments.
In an environment with air humidity of 8%, the reagent ball in example 1 is loaded into a chip body of a microfluidic chip, a detection sample is injected into the microfluidic chip, and then the detection is performed by using a portable automatic biochemical analyzer vp10 of shenzhen jinrui bio ltd, so as to detect a change value of absorbance at 546nm wavelength at 37 ℃. And the total serum protein concentration in the test sample can be calculated by using a calibrator provided by British Landau company for calibration.
1) And (3) testing precision: the microfluidic chip provided by the embodiment 1 of the invention is adopted to detect the detection sample 1# with the known serum total protein concentration, the serum total protein concentration of the detection sample 1# is 58g/L, and 20 detected concentration values are obtained after 20 times of detection.
The mean, standard deviation and coefficient of variation of the 20 concentration values were calculated to give a mean value of 59.74g/L, a standard deviation SD of 0.90 and a coefficient of variation CV of 1.51%.
It can be known that the measured concentration (59.74g/L) is very close to the actual concentration (58g/L), the accuracy is high, and the standard deviation and the coefficient of variation are small, which indicates that the stability of the reagent ball for measuring the total protein in the serum is good.
2) And (3) testing accuracy: by adopting the microfluidic chip provided by the embodiment 1 of the invention, a detection sample No. 2 with a known serum total protein concentration of 45g/L is tested, the detection is repeated three times to obtain concentration values, the average value of the concentration values measured 3 times is calculated to be 45.43g/L, and the relative deviation is 0.95%.
It can be seen that the measured concentration (45.43g/L) is close to the actual concentration (45g/L), the accuracy is high, and the relative deviation is small.
3) Clinical relevance analysis
Serum sample set a1 with different concentrations of total protein, i.e., a1 includes several serum samples with different concentrations of total protein, the total protein concentration of each serum sample in sample set a1 was measured using the microfluidic chip in example 1, and the total protein concentration of each serum sample in sample set a1 was measured using an Abaxis reagent disk. The detection results are shown in table 1, and for sample number 1 with the same concentration, the total serum protein concentration measured by using an Abaxis reagent disk is 58g/L, and the total serum protein concentration measured by using the microfluidic chip provided in the embodiment 1 of the invention is 56 g/L.
TABLE 1 test results of clinical relevance analysis
Taking the detection concentration value corresponding to the Abaxis reagent disk in the table 1 as the value of the X axis, taking the detection concentration value corresponding to the microfluidic chip as the value of the Y axis, and obtaining a correlation equation between two groups of detection results as follows:
Y＝0.9974x-0.4364；
the fit line of the correlation equation is shown in fig. 3, where the correlation coefficient R is 0.9908, and a closer correlation coefficient to 1 indicates a stronger correlation between the two sets of data. Therefore, the micro-fluidic chip provided by the embodiment of the invention has strong correlation with the test result of the Abaxis reagent disk.
4) Linear range test
The test method is as follows: serum samples at 6 dilution concentrations were mixed as shown in Table 2 using high concentration (activity) samples near the upper end of the linear range ([30, 100] g/L) and low concentration (activity) samples near the lower end of the linear range.
TABLE 2
| Sample numbering | 1 | 2 | 3 | 4 | 5 | 6 |
| High concentration (active) samples | 0 portion of | 1 part of | 2 portions of | 3 portions of | 4 portions of | 5 portions of |
| Low concentration (active) samples | 5 portions of | 4 portions of | 3 portions of | 2 portions of | 1 part of | 0 portion of |
The microfluidic chip provided by the embodiment 1 of the invention is adopted to respectively test the total protein concentration of 6 serum samples, each serum sample is tested for 3 times, and the average value (yi) of the concentration values measured by the total protein in the 6 serum samples is respectively calculated. The diluted concentration (xi) of each sample is taken as an independent variable, and the mean value (yi) of the concentration value measured by each sample is taken as a dependent variable to obtain a linear regression equation as follows:
Y＝1.0163x-0.5702；
the fit line of the correlation equation is shown in fig. 4, where the correlation coefficient R is 0.9998, and the closer the correlation coefficient R is to 1, the closer the result measured by using the microfluidic chip in the embodiment of the present invention is to the actual result after dilution is, i.e., the closer the measured value is to the actual value. Generally, when the kit detects a detection sample with TP concentration in the [30, 100] g/L interval, the linear correlation coefficient R is more than or equal to 0.990, and the requirement is met. The microfluidic chip provided by the embodiment of the invention has higher accuracy, and on the other hand, samples with different concentrations can be accurately measured, and the linear range is good.
5) Thermal stability test
The microfluidic chip of example 1 of the present invention was packaged in an environment with an air humidity of 8%, and stored in a dark environment at 37 ℃ for 0, 2, 3, 4, 6, and 8 days. The accuracy of the microfluidic chip in the embodiment of the invention is tested by taking Landau calibration products and quality control products as detection samples, and the relative deviation is within +/-10.0%. Specifically, two groups of quality control products (sample 1# and sample 2#) are provided by Landau corporation, the sample 1# and the sample 2# are respectively detected by adopting the microfluidic chip which meets the storage requirement, and the detection is carried out three times for the same type of microfluidic chip.
Table 3 shows the detection results of sample 1# detected by the microfluidic chip after storing each day, and table 4 shows the detection results of sample 2# detected by the microfluidic chip after storing each day. Wherein the average is the average of the concentrations detected in three times and the target value is the actual concentration of serum total protein in the sample.
As can be seen from tables 3 and 4, the microfluidic chip provided in the embodiment of the present invention has an absolute value of a relative deviation within 10.0% after being stored in an environment for 2, 3, 4, 6, and 8 days, and thus has good thermal stability, and can ensure accuracy of a detection result after being stored in an environment for a plurality of days.
TABLE 3 test results of sample No. 1
| Sample No. 1# | 1 | 2 | 3 | Mean value of | Target value (g/L) | Relative deviation of |
| Day 0 | 61.29 | 58.22 | 58.22 | 59.24 | 58.2 | 1.79％ |
| 2 days | 59.24 | 61.29 | 59.24 | 59.93 | 58.2 | 2.96％ |
| 3 days | 62.31 | 59.24 | 57.20 | 59.58 | 58.2 | 2.38％ |
| 4 days | 57.20 | 61.29 | 58.22 | 58.90 | 58.2 | 1.21％ |
| 6 days | 57.20 | 58.22 | 58.22 | 57.88 | 58.2 | -0.55％ |
| 8 days | 60.27 | 61.01 | 59.29 | 60.19 | 58.2 | 3.41％ |
TABLE 4 test results of sample No. 2
| Sample No. 2# | 1 | 2 | 3 | Mean value of | Target value (g/L) | Relative deviation of |
| Day 0 | 44.94 | 46.99 | 43.92 | 45.28 | 45 | 0.63％ |
| 2 days | 43.92 | 44.94 | 44.94 | 44.60 | 45 | -0.88％ |
| 3 days | 44.94 | 44.94 | 45.97 | 45.28 | 45 | 0.63％ |
| 4 days | 48.01 | 44.94 | 45.97 | 46.31 | 45 | 2.90％ |
| 6 days | 46.99 | 47.01 | 44.01 | 46.00 | 45 | 2.22％ |
| 8 days | 43.92 | 45.97 | 43.92 | 44.60 | 45 | -0.88％ |
6) Long term stability test
The microfluidic chip in example 1 of the present invention was packaged in an environment with an air humidity of 8%, and stored in a dark environment at 2-8 ℃ for 0, 3, 6, 9, 12, and 15 months. When the Landau calibrator and the quality control product are used as detection samples, the accuracy of the microfluidic chip is tested, and the relative deviation is within +/-10.0%. Specifically, two groups of quality control products (sample 3# and sample 4#) are provided by Landau corporation, the sample 3# and the sample 4# are detected by the microfluidic chip after being stored for 0, 3, 6, 9, 12 and 15 months in a dark environment at the temperature of 2-8 ℃, and the detection is carried out for three times on the same type of microfluidic chip.
Table 5 shows the results of the detection of sample 3# by the microfluidic chip after 0, 3, 6, 9, 12, and 15 months of storage in a light-shielding environment at 2-8 ℃, and table 6 shows the results of the detection of sample 4# by the microfluidic chip after 0, 3, 6, 9, 12, and 15 months of storage in a light-shielding environment at 2-8 ℃. Wherein the average is the average of the concentrations detected in three times and the target value is the actual concentration of serum total protein in the sample.
As can be seen from tables 5 and 6, the microfluidic chip provided in the embodiments of the present invention has an absolute value of the relative deviation within 10.0% after being stored in the environment for 0, 3, 6, 9, 12, and 15 months, so that the microfluidic chip has good long-term stability, and can ensure the accuracy of the detection result after being stored in the environment for a long time.
TABLE 5 examination result of sample No. 3
| Sample No. 3# | 1 | 2 | 3 | Mean value of | Target value (g/L) | Relative deviation of |
| 0 month | 59.66 | 58.65 | 57.63 | 58.65 | 58.6 | 0.08％ |
| 3 months old | 58.65 | 57.63 | 61.68 | 59.32 | 58.6 | 1.23％ |
| 6 months old | 59.66 | 60.67 | 58.65 | 59.66 | 58.6 | 1.80％ |
| 9 months old | 58.65 | 56.62 | 60.67 | 58.65 | 58.6 | 0.08％ |
| 12 months old | 56.62 | 57.63 | 60.67 | 58.31 | 58.6 | -0.50％ |
| 15 months old | 59.66 | 60.67 | 59.66 | 59.99 | 58.6 | 2.38％ |
TABLE 6 test results of sample No. 4
| Sample No. 4# | 1 | 2 | 3 | Mean value of | Target value (g/L) | Relative deviation of |
| 0 month | 44.91 | 46.91 | 43.81 | 45.21 | 44.9 | 0.68％ |
| 3 months old | 46.91 | 44.87 | 47.93 | 46.57 | 44.9 | 3.71％ |
| 6 months old | 44.87 | 44.87 | 44.87 | 44.87 | 44.9 | -0.07％ |
| 9 months old | 44.87 | 46.91 | 43.85 | 45.21 | 44.9 | 0.68％ |
| 12 months old | 47.93 | 45.73 | 44.87 | 46.17 | 44.9 | 2.83％ |
| 15 months old | 45.89 | 46.91 | 45.89 | 46.23 | 44.9 | 2.95％ |
7) Anti-interference capability
In order to verify the anti-interference capability of the reagent ball, the microfluidic chip in embodiment 1 of the present invention is used to test samples 5# -8 # mixed with other interfering substances. Wherein, the serum total protein concentration of sample No. 5 is 58.5g/L, contains ascorbic acid of 30mg/dl, the serum total protein concentration of sample No. 6 is 58.5g/L, contains bilirubin of 40mg/dl, the serum total protein concentration of sample No. 7 is 58.5g/L, contains hemoglobin of 400mg/dl, the serum total protein concentration of sample No. 8 is 58.5g/L, and contains triglyceride of 500 mg/dl.
TABLE 7 anti-interference test results
| Item type | Measured value | Relative deviation of |
| Sample No. 5 (sample containing 30mg/dl ascorbic acid 58.5 g/L) | 58.46 | -0.06％ |
| Sample No. 6 (bilirubin 40mg/dl 58.5g/L sample) | 59.62 | 1.91％ |
| Sample No. 7 (sample containing 400mg/dl hemoglobin 58.5 g/L) | 59.25 | 1.29％ |
| Sample No. 8 (sample containing 500mg/dl triglyceride 58.5 g/L) | 59.60 | 1.88％ |
From the anti-interference test results, the relative deviation of the detection results and the actual results is small and within the range of 2%, so that the micro-fluidic chip in the embodiment 1 of the invention can be proved to have better anti-interference capability, namely the reagent ball for measuring the total serum protein in the embodiment of the invention has better anti-interference capability.
In summary, the preparation method of the reagent ball for measuring total serum protein provided by the embodiment of the present invention is to mix a certain amount of buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, iodide, nonionic surfactant, stabilizer, excipient and water to form a mixed solution, then drop the mixed solution in liquid nitrogen in the form of liquid drops to form ice balls, and freeze-dry the ice balls to obtain the spherical reagent ball for measuring total serum protein. Wherein the mixed solution comprises the following components: 40-200mmol/L buffer solution, 10-40g/L blue vitriol, 20-80g/L potassium sodium tartrate, 10-40g/L iodide, 5-20g/L non-ionic surfactant, 0.1-10g/L stabilizer and 10-100g/L excipient. The sodium potassium tartrate provides an alkaline environment, so that copper ions in the blue-violet sulfate and peptide bonds of total proteins are subjected to a complex reaction to form a blue-violet complex, the color intensity of the blue-violet complex is combined to evaluate the content of the total proteins, the iodide is helpful for preventing the complex from being automatically reduced, the nonionic surfactant can increase the contact and combination of the total proteins and the copper ions of serum, and simultaneously can reduce the turbidity of the serum to a certain extent, so that the anti-interference effect is achieved, and the detection accuracy is improved; on the other hand, the excipient in the dosage range can ensure the shape and the re-melting solubility of the reagent ball, and is beneficial to completely freeze-drying the reagent ball. Namely, the reagent ball for measuring the total serum protein prepared by the method has better shape and meltdown solubility, and can be completely frozen and dried, thereby having higher stability and precision.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. A method for preparing a reagent ball for measuring total serum protein is characterized by comprising the following steps:
mixing buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, iodide, nonionic surfactant, stabilizer, excipient and water to form mixed solution;
dropping the liquid drops of the mixed liquid into liquid nitrogen to enable the liquid drops to form ice balls;
freeze-drying the ice ball to prepare the reagent ball for measuring the total serum protein;
wherein the mixed solution comprises the following components: 40-200mmol/L of buffer solution, 10-40g/L of copper sulfate pentahydrate, 20-80g/L of sodium potassium tartrate, 10-40g/L of iodide, 5-20g/L of nonionic surfactant, 0.1-10g/L of stabilizer and 10-100g/L of excipient, wherein the stabilizer comprises sodium carbonate and cholic acid.
2. The method for preparing reagent beads for measuring total serum protein according to claim 1, wherein said mixing buffer solution, copper sulfate pentahydrate, sodium potassium tartrate, iodide, nonionic surfactant, stabilizer, excipient and water to form a mixture solution comprises:
adding the buffer to a first predetermined amount of water;
after the buffer solution is completely dissolved in the water, sequentially adding copper sulfate pentahydrate, sodium potassium tartrate and iodide to form a first solution;
sequentially adding a nonionic surfactant, a stabilizer and an excipient into the first solution, and adding a second preset amount of water into the first solution to form a third preset amount of the mixed solution.
3. The method for preparing reagent beads for measuring total serum protein according to claim 1 or 2, wherein said excipient comprises at least one of mannitol, inositol, trehalose, PEG3350, PEG8000, polyvinylpyrrolidone, dextran 1 ten thousand, and dextran 4 ten thousand.
4. The method for preparing reagent beads for measuring total serum protein according to claim 1 or 2, wherein the buffer comprises at least one of sodium hydroxide buffer, potassium hydroxide buffer and disodium citrate buffer.
5. The method for preparing reagent balls for measuring total serum protein according to claim 1 or 2, wherein the non-ionic surfactant comprises at least one of polyethylene glycol octyl phenyl ether and tetrapolyethylene glycol monolauryl ether.
6. The method for preparing a reagent ball for measuring total serum protein according to claim 1 or 2, wherein the volume of said iceball is 2.5ul-3.5 ul.
7. A reagent ball prepared by the method for preparing a reagent ball for measuring total serum protein according to any one of claims 1 to 6.
8. A microfluidic chip comprising a chip body and the reagent ball of claim 7, wherein the reagent ball is disposed inside the chip body.
9. A biochemical analyzer comprising an analyzer body, a reaction chamber and the microfluidic chip of claim 8, wherein the reaction chamber is opened in the analyzer body, and the microfluidic chip is mounted in the reaction chamber.
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| CN114277088A (en) * | 2021-12-02 | 2022-04-05 | 深圳市锦瑞生物科技股份有限公司 | Total bilirubin determination reagent, preparation method of reagent ball and determination chip |
| CN114292898A (en) * | 2021-12-02 | 2022-04-08 | 深圳市锦瑞生物科技股份有限公司 | Preparation method of glutamic-oxalacetic transaminase determination reagent and reagent ball and determination chip |
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| CN114292898A (en) * | 2021-12-02 | 2022-04-08 | 深圳市锦瑞生物科技股份有限公司 | Preparation method of glutamic-oxalacetic transaminase determination reagent and reagent ball and determination chip |
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