CN117625733A - Lactic acid detection freeze-dried reagent ball, preparation method and microfluidic detection chip - Google Patents
Lactic acid detection freeze-dried reagent ball, preparation method and microfluidic detection chip Download PDFInfo
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 173
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 109
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 59
- 239000004310 lactic acid Substances 0.000 title claims abstract description 59
- 238000001514 detection method Methods 0.000 title claims abstract description 44
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims abstract description 28
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 claims abstract description 26
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 17
- 229940072107 ascorbate Drugs 0.000 claims abstract description 11
- 235000010323 ascorbic acid Nutrition 0.000 claims abstract description 11
- 239000011668 ascorbic acid Substances 0.000 claims abstract description 11
- 230000002452 interceptive effect Effects 0.000 claims abstract description 5
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 36
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- 239000000872 buffer Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 22
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 22
- 239000003381 stabilizer Substances 0.000 claims description 21
- 239000000243 solution Substances 0.000 claims description 20
- 239000007853 buffer solution Substances 0.000 claims description 19
- 239000008188 pellet Substances 0.000 claims description 18
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 14
- UZSCVCWALGRUTR-UHFFFAOYSA-N 4-amino-1,5-dimethyl-2-phenylpyrazol-3-one;hydron;chloride Chemical compound Cl.CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 UZSCVCWALGRUTR-UHFFFAOYSA-N 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 14
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 14
- 229930195725 Mannitol Natural products 0.000 claims description 14
- YNLCVAQJIKOXER-UHFFFAOYSA-N N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid Chemical compound OCC(CO)(CO)NCCCS(O)(=O)=O YNLCVAQJIKOXER-UHFFFAOYSA-N 0.000 claims description 14
- 239000000594 mannitol Substances 0.000 claims description 14
- 235000010355 mannitol Nutrition 0.000 claims description 14
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 13
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- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 13
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 9
- 239000000276 potassium ferrocyanide Substances 0.000 claims description 8
- 229940077386 sodium benzenesulfonate Drugs 0.000 claims description 8
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 8
- 108010015428 Bilirubin oxidase Proteins 0.000 claims description 7
- 239000007995 HEPES buffer Substances 0.000 claims description 7
- 229910052757 nitrogen Inorganic materials 0.000 claims description 7
- -1 potassium ferricyanide Chemical compound 0.000 claims description 7
- 239000002994 raw material Substances 0.000 claims description 7
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- 108010024957 Ascorbate Oxidase Proteins 0.000 claims description 5
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- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 abstract description 3
- 229930003268 Vitamin C Natural products 0.000 abstract description 3
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000006555 catalytic reaction Methods 0.000 description 3
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- 150000004060 quinone imines Chemical class 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- IULJSGIJJZZUMF-UHFFFAOYSA-N 2-hydroxybenzenesulfonic acid Chemical compound OC1=CC=CC=C1S(O)(=O)=O IULJSGIJJZZUMF-UHFFFAOYSA-N 0.000 description 2
- RLFWWDJHLFCNIJ-UHFFFAOYSA-N 4-aminoantipyrine Chemical compound CN1C(C)=C(N)C(=O)N1C1=CC=CC=C1 RLFWWDJHLFCNIJ-UHFFFAOYSA-N 0.000 description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 2
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 2
- 108010073450 Lactate 2-monooxygenase Proteins 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
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- 238000011088 calibration curve Methods 0.000 description 2
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
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- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- NMWCVZCSJHJYFW-UHFFFAOYSA-M sodium;3,5-dichloro-2-hydroxybenzenesulfonate Chemical compound [Na+].OC1=C(Cl)C=C(Cl)C=C1S([O-])(=O)=O NMWCVZCSJHJYFW-UHFFFAOYSA-M 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
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- LWKJNIMGNUTZOO-UHFFFAOYSA-N 3,5-dichloro-2-hydroxybenzenesulfonic acid Chemical compound OC1=C(Cl)C=C(Cl)C=C1S(O)(=O)=O LWKJNIMGNUTZOO-UHFFFAOYSA-N 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
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- 102000004877 Insulin Human genes 0.000 description 1
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- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a lactic acid detection freeze-dried reagent ball, a preparation method and a microfluidic detection chip, and belongs to the technical field of in-vitro detection. The lactic acid detection freeze-dried reagent ball divides a lactic acid detection reagent into two reagents, and is prepared into an R1 freeze-dried reagent ball and an R2 freeze-dried reagent ball, so that the stability of the reagent and the accuracy of the test are improved, and the storage time can be prolonged; in the R1 freeze-dried reagent ball, the ascorbate is used for eliminating the interference of vitamin C in blood on Trinder reaction, the bilirubin removing interfering agent is used for eliminating the interference of bilirubin, and the addition of the ascorbate and the bilirubin removing interfering agent enables the freeze-dried reagent ball to have stronger anti-interference capability and higher accuracy of detection results; the reagent R1 freeze-dried reagent ball and the reagent R2 freeze-dried reagent ball are particularly suitable for being packaged in a microfluidic detection chip, and the portable automatic biochemical analyzer is used for realizing in-vitro instant diagnosis of lactic acid.
Description
Technical Field
The invention relates to the technical field of in-vitro detection, belongs to the technical field of lactic acid detection, and in particular relates to a lactic acid detection freeze-dried reagent ball, a preparation method and a microfluidic detection chip.
Background
Lactic acid is an intermediate of sugar metabolism, mainly derived from skeletal muscle, brain, skin, renal medulla and erythrocytes. The concentration of lactic acid in the blood is related to the rate at which lactic acid is produced by these tissues and the metabolic rate of lactic acid by the liver, about 65% of which is metabolized by the liver. Elevated plasma lactate can be seen in physiological elevation due to intense exercise or dehydration, as well as pathological elevation due to: (1) Severe hypoxia of tissues occurs during shock, heart failure, hematopathy and pulmonary insufficiency, resulting in increased glycolysis of pyruvate to lactate, contributing to elevated lactate levels; (2) In some liver diseases, the clearance rate of the liver to lactic acid is reduced, so that the blood lactic acid can be increased; (3) The diabetes patients have absolute or/and relative deficiency of insulin, the organism can not effectively utilize blood sugar, and pyruvic acid is reduced into lactic acid in a large amount, so that lactic acid is accumulated in the body, and lactic acidosis occurs; (4) The administration of certain drugs or poisons (e.g., ethanol, methanol, salicylic acid, etc.) can also cause elevated blood lactic acid. Thus, measuring the concentration of lactic acid in plasma is of great importance for clinical diagnosis.
Examples of the method for measuring lactic acid include a chemical method, a gas chromatography method, an electrochemical method, an enzyme electrode induction method and an enzyme catalysis method. The chemical method has the advantages of complex operation, long time and poor accuracy. The gas chromatography has good accuracy and small sample size, but needs a gas chromatograph and cannot be used in a conventional laboratory. The electrochemical method has high sensitivity, wide linear range, rapidness, simplicity, convenience and accuracy, but special equipment is needed. The enzyme catalysis method has high sensitivity and wide linear range, is suitable for an automatic analyzer, and is an ideal common method for measuring lactic acid. The enzyme catalysis method can be classified into a lactate dehydrogenase method and a lactate oxidase method according to the reaction principle.
Lactate dehydrogenase methods include ultraviolet methods and colorimetric methods coupled with tetrazolium salts. The detection principle of the lactic acid oxidase method is as follows: firstly, under the action of lactic acid oxidase, lactic acid reacts with oxygen to generate pyruvic acid and hydrogen peroxide, and then under the action of peroxidase, hydrogen peroxide, 4-aminoantipyrine and 3, 5-dichloro-2-hydroxy benzenesulfonic acid react to generate quinone imine and water. The quinone imine is a red dye, the formation rate and the formation amount of the quinone imine are in direct proportion to the concentration of lactic acid, and the concentration of the lactic acid can be determined by detecting absorbance at a wavelength of 500-600 nm.
The lactic acid oxidase method has high sensitivity, good repeatability and wide linear range. However, the peroxidase has poor specificity to the substrate, serious interference reaction and poor analysis accuracy; lactic acid oxidase is unstable in a liquid reagent state and is easy to inactivate under the condition of higher temperature; moreover, the existing lactic acid detection reagent depends on a large-scale full-automatic biochemical analyzer in a laboratory, and is difficult to carry with it for immediate diagnosis.
Disclosure of Invention
In view of the above-mentioned defects or shortcomings in the prior art, the invention aims to provide a lactic acid detection freeze-dried reagent ball which has good stability and strong anti-interference capability and is convenient for instant diagnosis, a preparation method and a microfluidic detection chip.
The invention aims to provide a lactic acid detection freeze-dried reagent ball, which comprises an R1 freeze-dried reagent ball and an R2 freeze-dried reagent ball.
The R1 freeze-dried reagent ball comprises the following raw materials:
the R2 freeze-dried reagent ball comprises the following raw materials:
preferably, the first buffer and the second buffer each independently comprise one or more of phosphate buffer, tris buffer, N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer or 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES) buffer.
Preferably, the first stabilizer and the second stabilizer each independently comprise one or more of sucrose, fructose, glycerol or bovine serum albumin.
Preferably, the first excipient and the second excipient each independently comprise one or more of mannitol, inositol, PEG3350, PEG8000, dextran 1 ten thousand, or dextran 4 ten thousand.
Preferably, the pH of the reagents R1 and R2 are each independently 6 to 9.
Preferably, the bilirubin-interfering agent comprises one or more of potassium ferrocyanide, potassium ferricyanide or bilirubin oxidase.
The second object of the invention is to provide a preparation method of lactic acid detection freeze-dried reagent balls, which comprises the following steps:
firstly, preparing a reagent R1 and a reagent R2 respectively, then forming the reagent R1 and the reagent R2 into ice balls respectively, and then freeze-drying the ice balls of the reagent R1 and the reagent R2 respectively.
Preferably, the preparation method of the reagent R1 comprises the following steps:
firstly, adding a first buffer solution into water, fully dissolving, then adding 3, 5-dichloro-2-hydroxy sodium benzenesulfonate and bilirubin removing interference agent, then regulating the pH value of the solution, then sequentially adding a first stabilizer and a first excipient, then adding ascorbate and peroxidase, and then carrying out volume fixing on the solution.
Preferably, the preparation method of the reagent R2 comprises the following steps:
firstly, adding a second buffer solution into water, fully dissolving, then adding 4-aminoantipyrine hydrochloride, regulating the pH value of the solution, then sequentially adding a second stabilizer and a second excipient, and then adding lactic acid oxidase, and then, carrying out constant volume on the solution.
Preferably, the reagent R1 and the reagent R2 are directly dripped into liquid nitrogen to form ice balls through a quantitative control bead dispenser respectively.
Preferably, the reagent R1 ice ball and the reagent R2 ice ball are lyophilized in a vacuum freeze dryer, respectively.
Preferably, the volume of the R1 lyophilized reagent pellet and the R2 lyophilized reagent pellet are each independently 3.0 to 4.0. Mu.L.
The invention further aims to provide a microfluidic detection chip, which comprises the lactic acid detection freeze-dried reagent ball.
Preferably, the lactic acid detecting reagent balls are packaged in a microfluidic chip in an environment with an air humidity of 8%.
The beneficial effects of the invention include:
the lactic acid detection reagent is divided into two parts, and the R1 freeze-dried reagent ball and the R2 freeze-dried reagent ball are prepared, so that the stability of the reagent and the accuracy of the test are improved, and the storage time can be prolonged; in the R1 freeze-dried reagent ball, the ascorbate is used for eliminating the interference of vitamin C in blood on Trinder reaction, the bilirubin removing interfering agent is used for eliminating the interference of bilirubin, and the addition of the ascorbate and the bilirubin removing interfering agent enables the freeze-dried reagent ball to have stronger anti-interference capability; 3, 5-dichloro-2-hydroxy sodium benzenesulfonate is added into the R1 freeze-dried reagent ball, and 4-aminoantipyrine hydrochloride is added into the R2 freeze-dried reagent ball, so that the reaction of 3, 5-dichloro-2-hydroxy sodium benzenesulfonate and 4-aminoantipyrine hydrochloride before detection is avoided, and in addition, the reaction of peroxidase in the R1 freeze-dried reagent ball and 4-aminoantipyrine hydrochloride is also avoided, and the accuracy of the test is further ensured. In addition, the R1 freeze-dried reagent balls and the R2 freeze-dried reagent balls are particularly suitable for being packaged in a microfluidic detection chip, and the in-vitro instant diagnosis of lactic acid is facilitated by utilizing the portable automatic biochemical analyzer.
Detailed Description
In the following description, certain specific details are included to provide a thorough understanding of various disclosed embodiments. One skilled in the relevant art will recognize, however, that the embodiments may be practiced without one or more of the specific details, or with other methods, components, materials, etc.
Throughout the specification and the claims which follow, unless the context requires otherwise, the words "comprise" and "comprising" are to be interpreted in an open-ended, inclusive sense, i.e. "including but not limited to.
Reference throughout this specification to "one embodiment" or "an embodiment" or "one preferred embodiment" or "certain embodiments" means that a particular reference element, structure, or feature described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" or "in a preferred embodiment" or "in certain embodiments" appearing in various places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular elements, structures, or features may be combined in any suitable manner in one or more embodiments.
According to a first aspect of the invention, there is provided a lactic acid detection freeze-dried reagent pellet comprising an R1 freeze-dried reagent pellet and an R2 freeze-dried reagent pellet.
The R1 freeze-dried reagent ball comprises the following raw materials in concentration:
the R2 freeze-dried reagent balls comprise the following raw materials in concentration:
in the present invention, the concentration of the first buffer solution in the reagent R1 is, for example, 20mmol/L, 40mmol/L, 50mmol/L, 60mmol/L, 80mmol/L, 100mmol/L, 120mmol/L, 140mmol/L, 160mmol/L, 180mmol/L or 200mmol/L.
In reagent R1, the concentration of sodium 3, 5-dichloro-2-hydroxybenzenesulfonate is, for example, 2g/L, 4g/L, 6g/L, 8g/L, 10g/L, 12g/L, 14g/L, 16g/L, 18g/L, or 20g/L.
In the reagent R1, the concentration of peroxidase is, for example, 20KU/L, 40KU/L, 60KU/L, 80KU/L, 100KU/L, 120KU/L, 160KU/L, 180KU/L or 200KU/L.
In the reagent R1, the concentration of the ascorbate is, for example, 20KU/L, 40KU/L, 60KU/L, 80KU/L, 100KU/L, 120KU/L, 160KU/L, 180KU/L or 200KU/L.
In the invention, the ascorbate is used for eliminating the interference of vitamin C in blood on Trinder reaction, so that the detection accuracy is improved.
In reagent R1, the concentration of the bilirubin-interfering agent is, for example, 0.01g/L, 0.1g/L, 0.2g/L, 0.3g/L, 0.4g/L, 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L or 1g/L.
In the invention, the bilirubin interference removing agent is used for eliminating the interference of bilirubin in blood and improving the detection accuracy.
In reagent R1, the concentration of the first stabilizer is, for example, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L or 50g/L.
In reagent R1, the concentration of the first excipient is, for example, 20g/L, 40g/L, 60g/L, 80g/L, 100g/L, 120g/L, 140g/L, 160g/L, 180g/L or 200g/L.
In the present invention, the concentration of the second buffer in the reagent R2 is, for example, 20mmol/L, 40mmol/L, 50mmol/L, 60mmol/L, 80mmol/L, 100mmol/L, 120mmol/L, 140mmol/L, 160mmol/L, 180mmol/L or 200mmol/L.
In reagent R2, the concentration of 4-aminoantipyrine hydrochloride is, for example, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L or 10g/L.
In the reagent R2, the concentration of the lactate oxidase is, for example, 0.1KU/L, 0.2KU/L, 0.4KU/L, 0.6KU/L, 0.8KU/L, 1KU/L, 1.2KU/L, 1.4KU/L, 1.6KU/L, 1.8KU/L or 2KU/L.
In reagent R2, the concentration of the second stabilizer is, for example, 5g/L, 10g/L, 15g/L, 20g/L, 25g/L, 30g/L, 35g/L, 40g/L, 45g/L or 50g/L.
In reagent R2, the concentration of the second excipient is, for example, 20g/L, 40g/L, 60g/L, 80g/L, 100g/L, 120g/L, 140g/L, 160g/L, 180g/L or 200g/L.
In the invention, 3, 5-dichloro-2-hydroxy sodium benzenesulfonate and 4-aminoantipyrine hydrochloride, as well as peroxidase and 4-aminoantipyrine hydrochloride have certain reactivity. Thus, sodium 3, 5-dichloro-2-hydroxybenzenesulfonate and peroxidase are packaged separately from 4-aminoantipyrine hydrochloride in two different reagents. Namely, 3, 5-dichloro-2-sodium hydroxybenzenesulfonate and peroxidase are added into the R1 freeze-dried reagent ball, and 4-aminoantipyrine hydrochloride is added into the R2 freeze-dried reagent ball, so that the reaction of 3, 5-dichloro-2-sodium hydroxybenzenesulfonate and peroxidase with 4-aminoantipyrine hydrochloride before detection is avoided, and the accuracy of the test is further ensured.
In addition, the reagent R1 and the reagent R2 are prepared into the form of freeze-dried pellets, so that the stability and portability of the reagent are improved, the reagent is particularly suitable for packaging the R1 freeze-dried reagent pellets and the R2 freeze-dried reagent pellets in a microfluidic detection chip, and the in-vitro instant diagnosis of lactic acid is realized by using a portable automatic biochemical analyzer.
In a specific application scenario, when a detection sample flows into a detection chip during diagnosis, the detection sample dissolves the R1 freeze-dried reagent balls and the R2 freeze-dried reagent balls, and the following reaction is performed:
in a preferred embodiment of the present invention, the first buffer and the second buffer each independently comprise one or more of phosphate buffer, tris buffer, N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer or 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES) buffer.
In the present invention, the first buffer is, for example, a phosphate buffer, a Tris buffer, an N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer, a 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES) buffer, a phosphate buffer and a Tris buffer, a phosphate buffer and an N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer, or a combination of a phosphate buffer, a Tris buffer and a 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES) buffer.
In the present invention, the second buffer is, for example, a phosphate buffer, a Tris buffer, an N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer, a 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES) buffer, a phosphate buffer and a Tris buffer, a phosphate buffer and an N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer, or a combination of a phosphate buffer, a Tris buffer and a 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES) buffer.
In a preferred embodiment of the present invention, each of the first and second stabilizers independently comprises one or more of sucrose, fructose, glycerol or bovine serum albumin.
In the present invention, the first stabilizer is, for example, sucrose, fructose, glycerol, bovine serum albumin, sucrose and fructose, sucrose, fructose and glycerol, glycerol and bovine serum albumin, or a combination of fructose, glycerol and bovine serum albumin.
In the present invention, the second stabilizer is, for example, sucrose, fructose, glycerol, bovine serum albumin, sucrose and fructose, sucrose, fructose and glycerol, glycerol and bovine serum albumin, or a combination of fructose, glycerol and bovine serum albumin.
In a preferred embodiment of the present invention, the first excipient and the second excipient each independently comprise one or more of mannitol, inositol, PEG3350, PEG8000, dextran 1 ten thousand, or dextran 4 ten thousand.
In the present invention, the first excipient is, for example, mannitol, inositol, PEG3350, PEG8000, dextran 1 ten thousand, dextran 4 ten thousand, mannitol and inositol, mannitol and PEG3350, PEG8000 and dextran 1 ten thousand, inositol and dextran 4 ten thousand, mannitol, inositol and PEG3350, or a combination of mannitol, inositol, PEG8000 and dextran 1 ten thousand.
In the present invention, the second excipient is, for example, mannitol, inositol, PEG3350, PEG8000, 1 million of glucan, 4 million of glucan, mannitol and inositol, mannitol and PEG3350, PEG8000 and 1 million of glucan, 4 million of inositol and glucan, mannitol, inositol and PEG3350, or a combination of mannitol, inositol, PEG8000 and 1 million of glucan.
In a preferred embodiment of the present invention, the pH values of the reagents R1 and R2 are each independently 6 to 9.
In the present invention, the pH of the reagent R1 is, for example, 6, 6.3, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.3, 8.5, 8.7 or 9.
In the present invention, the pH of the reagent R2 is, for example, 6, 6.3, 6.5, 6.8, 7, 7.2, 7.5, 7.8, 8, 8.3, 8.5, 8.7 or 9.
In a preferred embodiment of the present invention, the bilirubin-interfering agent comprises one or more of potassium ferrocyanide, potassium ferricyanide, or bilirubin oxidase.
In the present invention, the bilirubin-disturbing agent is, for example, potassium ferrocyanide, potassium ferricyanide, bilirubin oxidase, potassium ferrocyanide and potassium ferricyanide, potassium ferrocyanide and bilirubin oxidase, potassium ferricyanide and bilirubin oxidase, or a combination of potassium ferrocyanide, potassium ferricyanide and bilirubin oxidase.
According to a second aspect of the present invention, there is provided a method for preparing a lactic acid detecting freeze-dried reagent pellet, the method comprising:
firstly, preparing a reagent R1 and a reagent R2 respectively, then forming the reagent R1 and the reagent R2 into ice balls respectively, and then freeze-drying the ice balls of the reagent R1 and the reagent R2 respectively.
In a preferred embodiment of the present invention, the preparation method of the reagent R1 comprises:
firstly, adding a first buffer solution into water, fully dissolving, then adding 3, 5-dichloro-2-hydroxy sodium benzenesulfonate and bilirubin removing interference agent, then regulating the pH value of the solution, then sequentially adding a first stabilizer and a first excipient, then adding ascorbate and peroxidase, and then carrying out volume fixing on the solution.
In a preferred embodiment of the present invention, the method for preparing reagent R2 comprises:
firstly, adding a second buffer solution into water, fully dissolving, then adding 4-aminoantipyrine hydrochloride, regulating the pH value of the solution, then sequentially adding a second stabilizer and a second excipient, and then adding lactic acid oxidase, and then, carrying out constant volume on the solution.
In a preferred embodiment of the invention, the reagent R1 and the reagent R2 are respectively directly dripped into liquid nitrogen to form ice balls through a quantitative control bead dispenser.
In a preferred embodiment of the present invention, the reagent R1 ice ball and the reagent R2 ice ball are freeze-dried in a vacuum freeze-dryer, respectively.
In a preferred embodiment of the present invention, the volume of the reagent R1 lyophilized reagent pellet and the reagent R2 lyophilized reagent pellet are each independently 3.0 to 4.0. Mu.L.
Specifically, the preparation method of the lactic acid detection freeze-dried reagent ball comprises the following steps:
s1, respectively preparing a reagent R1 and a reagent R2.
The preparation method of the reagent R1 comprises the following steps:
a. distilled water is added into a beaker;
b. b, weighing the buffer solution component, adding the buffer solution component into a beaker in the step a, fully dissolving the buffer solution component, and then adding 3, 5-dichloro-2-hydroxy sodium benzenesulfonate and bilirubin interference removing agent;
c. b, after the step b is finished, regulating the pH value of the solution;
d. after the pH is regulated, sequentially adding a stabilizer and an excipient into the beaker;
e. after the step d is finished, adding ascorbate and peroxidase;
f. after all the reagents are added through the steps, the solution is subjected to constant volume and shaking for standby.
The preparation method of the reagent R2 comprises the following steps:
a. distilled water is added into a beaker;
b. b, weighing a buffer solution component, adding the buffer solution component into a beaker in the step a, and adding 4-aminoantipyrine hydrochloride after the buffer solution component is fully dissolved;
c. b, after the step b is finished, regulating the pH value of the solution;
d. after the pH is regulated, sequentially adding a stabilizer and an excipient into the beaker;
e. after the step d is finished, adding lactic acid oxidase;
f. after all the reagents are added through the steps, the solution is subjected to constant volume and shaking for standby.
S2, respectively and directly dripping the reagent R1 and the reagent R2 into liquid nitrogen through a quantitative control bead dispenser to form ice balls.
S3, respectively placing the reagent R1 ice ball and the reagent R2 ice ball in a vacuum freeze dryer for freeze drying.
S4, nitrogen repressing, and collecting and storing the freeze-dried reagent balls in a dry aluminum bottle for later use, wherein the volume of the freeze-dried reagent balls is 3.0-4.0 mu L.
According to a third aspect of the present invention, there is provided a microfluidic detection chip comprising lactic acid detection freeze-dried reagent spheres as described above.
In a preferred embodiment of the invention, one reagent R1 lyophilized reagent pellet and one reagent R2 lyophilized reagent pellet are packaged in one microfluidic chip in an environment with an air humidity of 8%.
When the kit is used, a microfluidic detection chip for packaging a reagent R1 freeze-dried reagent ball and a reagent R2 freeze-dried reagent ball is added into a sample to be detected, and then the sample is put into a portable automatic biochemical analyzer to detect the light absorption value of 505nm/600nm wavelength at 37 ℃; and then calculating the lactic acid content in the sample by using a calibration curve.
Examples
The present invention will be described in further detail with reference to examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
In the following examples, each raw material component was a commercially available product unless otherwise specified.
Phosphate buffer is used for the first buffer and the second buffer;
the first stabilizer and the second stabilizer are glycerol;
mannitol is used as the first excipient and the second excipient;
potassium ferrocyanide is used as bilirubin-eliminating disturbing agent.
The contents of the respective components in examples 1 to 5 and comparative examples 1 to 3 are shown in Table 1.
TABLE 1 content of each component in examples 1 to 5 and comparative examples 1 to 3
S1 reagents R1 and R2 were prepared as follows.
First, reagent R1 was prepared:
a. distilled water is added into a beaker;
b. b, weighing the buffer solution component, adding the buffer solution component into a beaker in the step a, fully dissolving the buffer solution component, and then adding 3, 5-dichloro-2-hydroxy sodium benzenesulfonate and bilirubin interference removing agent;
c. b, after the step b is finished, regulating the pH value of the solution;
d. after the pH is regulated, sequentially adding a stabilizer and an excipient into the beaker;
e. after the step d is finished, adding ascorbate and peroxidase;
f. after all the reagents are added through the steps, the solution is subjected to constant volume and shaking for standby.
Then, reagent R2 was prepared:
a. distilled water is added into a beaker;
b. b, weighing a buffer solution component, adding the buffer solution component into a beaker in the step a, and adding 4-aminoantipyrine hydrochloride after the buffer solution component is fully dissolved;
c. b, after the step b is finished, regulating the pH value of the solution;
d. after the pH is regulated, sequentially adding a stabilizer and an excipient into the beaker;
e. after the step d is finished, adding lactic acid oxidase;
f. after all the reagents are added through the steps, the solution is subjected to constant volume and shaking for standby.
S2, respectively and directly dripping the reagent R1 and the reagent R2 into liquid nitrogen through a quantitative control bead dispenser to form ice balls.
S3, respectively placing the reagent R1 ice ball and the reagent R2 ice ball in a vacuum freeze dryer for freeze drying.
S4, nitrogen repressing, and collecting and storing the freeze-dried reagent balls in a dry aluminum bottle for later use, wherein the volume of the freeze-dried reagent balls is 3.0-4.0 mu L.
S5, in the environment with the air humidity of 8%, packaging one reagent R1 freeze-dried reagent ball and one reagent R2 freeze-dried reagent ball into one microfluidic chip.
When the kit is used, a microfluidic detection chip for encapsulating the reagent R1 freeze-dried reagent balls and one reagent R2 freeze-dried reagent ball is added into a sample to be detected, and then the sample is put into a portable automatic biochemical analyzer to detect the light absorption value of 505nm/600nm wavelength at 37 ℃; and then calculating the lactic acid content in the sample by using a calibration curve.
Samples were tested using the microfluidic test chips obtained in examples 1 to 5 and comparative examples 1 to 3. The test results are shown in Table 2.
TABLE 2 lactic acid detection concentrations of examples 1 to 5 and comparative examples 1 to 3
The above is a reproducibility test of the quality control product, and the test result of example 2 is preferable (pH 7.5), and the dispersion coefficient CV is high in comparative example 1 because it does not contain the bilirubin-interfering agent.
Claims (10)
1. The lactic acid detection freeze-dried reagent ball is characterized by comprising an R1 freeze-dried reagent ball and an R2 freeze-dried reagent ball;
the R1 freeze-dried reagent ball comprises the following raw materials:
the R2 freeze-dried reagent ball comprises the following raw materials:
2. the lactic acid detecting freeze-dried reagent pellet of claim 1, wherein the first buffer and the second buffer each independently comprise one or more of phosphate buffer, tris buffer, N-Tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS) buffer, or 4- (2-hydroxyethyl) -1-piperazine ethane sulfonic acid (HEPES) buffer.
3. The lactic acid detecting freeze-dried reagent pellet of claim 1, wherein the first stabilizer and the second stabilizer each independently comprise one or more of sucrose, fructose, glycerol, or bovine serum albumin.
4. The lactic acid detecting freeze-dried reagent pellet of claim 1, wherein the first excipient and the second excipient each independently comprise one or more of mannitol, inositol, PEG3350, PEG8000, dextran 1 ten thousand, or dextran 4 ten thousand.
5. The lactic acid detecting freeze-dried reagent ball according to claim 1, wherein the pH values of the reagent R1 and the reagent R2 are each independently 6 to 9.
6. The lactic acid detecting freeze-dried reagent ball of claim 1, wherein the bilirubin-disturbing agent comprises one or more of potassium ferrocyanide, potassium ferricyanide, or bilirubin oxidase.
7. The method for preparing a freeze-dried reagent ball for lactic acid assay according to claims 1 to 6, wherein the reagent R1 and the reagent R2 are prepared first, then the reagent R1 and the reagent R2 are formed into ice balls, respectively, and then the reagent R1 ice balls and the reagent R2 ice balls are freeze-dried, respectively.
8. The method for preparing the lactic acid detecting freeze-dried reagent ball according to claim 7, wherein:
the preparation method of the reagent R1 comprises the following steps:
firstly, adding a first buffer solution into water, fully dissolving, then adding 3, 5-dichloro-2-hydroxy sodium benzenesulfonate and bilirubin removing interfering agent, then adjusting the pH value of the solution, then sequentially adding a first stabilizer and a first excipient, then adding ascorbate and peroxidase, and then carrying out volume fixing on the solution;
the preparation method of the reagent R2 comprises the following steps:
firstly, adding a second buffer solution into water, fully dissolving, then adding 4-aminoantipyrine hydrochloride, regulating the pH value of the solution, then sequentially adding a second stabilizer and a second excipient, and then adding lactic acid oxidase, and then, carrying out constant volume on the solution.
9. The method for preparing the lactic acid detection freeze-dried reagent ball according to claim 7, wherein the reagent R1 and the reagent R2 are directly dripped into liquid nitrogen to form ice balls through a quantitative control bead dispenser;
preferably, the reagent R1 ice ball and the reagent R2 ice ball are respectively freeze-dried in a vacuum freeze dryer;
preferably, the volume of the R1 lyophilized reagent pellet and the R2 lyophilized reagent pellet are each independently 3.0 to 4.0. Mu.L.
10. A microfluidic detection chip, comprising the lactic acid detection freeze-dried reagent ball according to claims 1-6;
preferably, the lactic acid detecting reagent balls are packaged in a microfluidic chip in an environment with an air humidity of 8%.
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