Reagent for determining ionized calcium in blood sample, biochemical analysis chip and determination method

Abstract

The invention discloses a reagent for determining ionized calcium in a blood sample, the reagent comprises a blood sample ionized calcium determination reagent and a diluent, the blood sample ionized calcium determination reagent comprises phospholipase D [EC 3.1. 4.4], choline oxidase [1.1. 3.17], peroxidase [EC 1.11.1.7], a first color developing agent, a divalent metal salt, a first buffer agent, a first additive and a cryoprotectant; the diluent comprises phospholipid, a second color developing agent, a surfactant, a second additive and a second buffering agent; the blood sample ionized calcium determination reagent exists in the form of freeze-dried reagent balls, and the freeze-dried reagent balls are prepared by freeze-drying liquid drops of the blood sample ionized calcium determination reagent. The invention also discloses a corresponding biochemical analysis chip and a determination method, the biochemical analysis chip comprises a chip body and a reagent for determining ionized calcium in a blood sample, the chip body is provided with a colorimetric hole and a diluent tank, the freeze-dried reagent ball is accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank.

Description

Technical Field

[0001] The invention relates to the technical field of plasma ion calcium determination, and in particular to a reagent, a biochemical analysis chip and a determination method for determining ion calcium in a blood sample. Background Art

[0002] Calcium ions are indispensable ions for various physiological activities of the body. They play an important role in maintaining the bioelectric potential on both sides of the cell membrane and maintaining normal nerve conduction function. In the field of clinical examination, the detection and quantitative analysis of calcium ions in body fluids such as blood and urine are often used to diagnose endocrine diseases such as parathyroid abnormalities, circulatory system diseases such as hypertension and arteriosclerosis, etc. At the same time, calcium is also one of the factors that play a role in the mechanism of blood coagulation, which is of great significance in clinical treatment and medication.

[0003] Calcium in the blood can be divided into three types: diffusible ionized calcium, which is the physiologically active part; diffusible non-ionized calcium, which is complexed calcium combined with citric acid and bicarbonate; non-diffusible calcium, which is calcium combined with protein. The determination methods of blood calcium are generally divided into total calcium determination and ionized calcium determination. Common determination methods for serum total calcium include colorimetry, flame photometry, atomic absorption spectrophotometry, titration, and radionuclide dilution mass spectrometry. The measurement of ionized calcium in blood or serum is mostly carried out clinically using the ion selective electrode method (ISE). Although the ISE method has good accuracy and high precision, the electrode loss of this method is large, the failure rate is high and needs to be replaced regularly, the operation time is long, and a large number of samples cannot be processed at one time.

[0004] Enzymatic assays utilizing the activation or inhibition of enzymes by calcium can process a large number of samples at once. For example, JP-A-62-195297 discloses an analytical composition utilizing the activation of phospholipase D by calcium ions; JP-A-62-36199 discloses an analytical composition utilizing the activation of calmodulin by calcium and the activation of calmodulin-dependent enzymes by the activated calmodulin. JP-A-1-231896 discloses an analytical composition utilizing calcium ions to activate phospholipase A2. JP-A-2-142498 proposes an analytical composition utilizing the inhibitory effect of calcium ions on pyruvate kinase. JP-A-2-276597 proposes an assay method utilizing the activation of amylase by calcium ions. JP-A-4-23999 proposed a reagent composed of phospholipids, phospholipase D, choline oxidase, surfactant and divalent metal salt, which broadened the detection limit of calcium ions, eliminated the interference of salts or other metal ions present in body fluid samples, improved the reagent performance, and made continuous analysis possible.

[0005] The traditional enzymatic method has a narrow analysis range for ionized calcium, is easily affected by coexisting substances, lacks reproducibility, and the traditional liquid detection reagent has defects such as cumbersome detection steps, complicated operation or expensive detection instruments. For example, the method using calmodulin (JP-A-62-36199) has the following disadvantages: calmodulin is difficult to obtain, the measurable range of calcium concentration is narrow so that the sample needs to be diluted, and the two-stage activation system requires a long reaction time. The method using phospholipase A2 (JP-A-1-231896) and the method using pyruvate kinase (JP-A-2-142498) will produce errors when applied to serum determination because these enzymes are present in blood. In addition, the method using pyruvate kinase is less accurate because it utilizes the enzyme inhibition of calcium ions. In addition, the method using amylase (JP-A-2-276597) has problems in the reproducibility of the measured value because the blank reaction is too large. The method using phospholipase D (JP-A-62-195297) can accurately complete the measurement because the enzyme used is not present in the blood, but it is still affected by the coexisting salts. In addition, it is not suitable for continuous measurement. The method of JP-A-4-23999 adds surfactants and divalent metal salts while using phospholipase D to improve the specificity of the reagent. However, the above methods are all traditional liquid detection reagents, which have the defects of complex reaction process, multi-step reaction, cumbersome operation, low test efficiency, high reagent and instrument costs, etc. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art. In view of the problems in the related art that the analysis range is narrow, it is easy to be affected by coexisting substances, lacks reproducibility, has cumbersome detection steps, complicated operations, and is time-consuming, a reagent, a biochemical analysis chip, and a determination method for determining ionized calcium in blood samples are provided, and a favorable detection method for ionized calcium that has never been developed in the field of clinical examination is provided. The determination method is fast, convenient, stable, and has good specificity. It can quantitatively determine the calcium ion level in body fluids with high precision through simple operations; the addition of divalent metal salts and surfactants greatly broadens the detection limit of calcium ions and eliminates the influence of salts or other metal ions present in body fluid samples; in addition, the data analysis system of the present invention is also suitable for automatic analysis devices.

[0007] The first aspect of the present invention is to provide a reagent for measuring blood sample ion calcium, comprising: a blood sample ion calcium measuring reagent and a diluent, wherein the blood sample ion calcium measuring reagent comprises the following components: phospholipase D [EC3.1.4.4], choline oxidase [1.1.3.17], peroxidase [EC1.11.1.7], a first color developer, a divalent metal salt, a first buffer, a first additive and a cryoprotectant; the diluent comprises the following components: phospholipids, a second color developer, a surfactant, a second additive and a second buffer; the blood sample ion calcium measuring reagent exists in the form of a freeze-dried reagent ball, and the freeze-dried reagent ball is made by freeze-drying droplets of the blood sample ion calcium measuring reagent.

[0008] Preferably, the sources of the phospholipase D [EC3.1.4.4] include: animal sources, plant sources or microbial sources; the phospholipase D [EC3.1.4.4] includes: phospholipase D prepared by integrating microbial genes into microorganisms through genetic recombination technology and phospholipase D prepared by microorganisms with improved characteristics through genetic modification; the concentration of the phospholipase D [EC3.1.4.4] is 0.0001-10U / ml.

[0009] Preferably, the choline oxidase [1.1.3.17] comprises: choline oxidase of microbial origin; the microbial source comprises Arthrobacter sphaeroides or Alcaligenes sphaeroides; the concentration of the choline oxidase is 0.1-10 U / ml.

[0010] Preferably, the peroxidase [EC1.11.1.7] comprises peroxidase obtained from horseradish; and the concentration of the peroxidase [EC1.11.1.7] is 0.1-300 U / ml.

[0011] Preferably, the first developer comprises one or more of 4-aminoantipyrine (4-AA) and 3-methylbenzothiazolinonehydrazone (MBTH); and the concentration of the first developer is 0.001-10 wt %.

[0012] Preferably, the divalent metal salt includes one or more of a divalent alkaline earth metal salt or a divalent transition metal salt; the divalent alkaline earth metal salt or the divalent transition metal salt includes a chloride or sulfate soluble in an aqueous solution; and the concentration of the divalent metal salt is 0.1-200 mmol / L.

[0013] Preferably, the first buffer includes a buffer capable of buffering to a pH value in the range of 5 to 9; including: one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 2-morpholineethanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(trihydroxymethyl)methane; the concentration of the first buffer is 1-100 mmol / L to adjust the pH value to the range of 4.0 to 10.0 or the concentration of the first buffer is 10-500 mmol / L to adjust the pH value to the range of 6.0 to 9.0.

[0014] Preferably, the first additive includes a monovalent metal salt, a sugar, a sugar alcohol or a protein, and also includes a chelating agent, an amine, a basic amino acid or a heterocyclic compound; the monovalent metal salt includes chloride, sulfate or acetate of sodium or potassium; the sugar includes sucrose or lactose; the sugar alcohol includes sorbitol or mannitol; the protein includes bovine serum albumin; and the concentration of the first additive is 0.1-30wt%.

[0015] Preferably, the cryoprotectant acts as a stabilizer and an excipient at the same time, and the cryoprotectant includes sugars, polyols, amino acids, proteins and peptide excipients, and cyclic oligosaccharide-based polymers; wherein, The sugars include one or more of glucose, sucrose, lactose, maltose and trehalose; The polyols include one or more of mannitol, inositol, glycerol, ethylene glycol and polyethylene glycol; The amino acids include trimethylglycine, glycine, arginine or any salt thereof; The protein and peptide excipients include one or more of mucopolysaccharide protein, casein and bovine serum albumin; The cyclic oligosaccharide-based polymer comprises α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether, or any derivatives thereof, and any combination thereof; the concentration of the cryoprotectant is 0.1-60 wt %.

[0016] Preferably, the phospholipid comprises lysophosphatidylcholine or phosphatidylcholine; the concentration of the phospholipid is 0.2-100 mg / ml.

[0017] Preferably, the second developer includes 2,4,6-tribromo-3-hydroxybenzoic acid, sodium 3,5-dichloro-2-hydroxybenzenesulfonate, phenol, toluidine, aniline and derivatives of these compounds; aniline derivatives include N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, N-ethyl-N-(2-carboxy-3-sulfopropyl)-3 -methoxyaniline sodium salt, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt, N-ethyl-N-(3-sulfopropyl)aniline sodium salt, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3'5-dimethoxyaniline sodium salt, N'N-di(4-sulfobutyl)-3,5-dimethylaniline disodium salt (MADB) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt; the concentration of the second developer is 0.001-10wt%.

[0018] Preferably, the surfactant includes a nonionic surfactant and an anionic surfactant; the nonionic surfactant includes polyethylene glycol monooctylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, polyoxyethylene lauryl ether, polyoxyethylene cetyl ester, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate (Span40), Tween 20, Tween 40, Tween 80 and Tween 60; the anionic surfactant includes sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate and sodium lauryl sarcosine; the nonionic surfactant and the anionic surfactant are used alone or in combination of two or more; the concentration of the surfactant is 0.001-10wt%.

[0019] Preferably, the second buffer is a buffer capable of buffering to a pH value in the range of 5 to 8; including: one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 2-morpholineethanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane); the concentration of the second buffer is 1 to 1000 mmol / L to adjust the pH value to 4.0-10 or the concentration of the second buffer is 10 to 100 mmol / L to adjust the pH value to 5.0-8.0.

[0020] The second aspect of the present invention is to provide a biochemical analysis chip for measuring ion calcium in blood samples. The biochemical analysis chip includes a chip body and a reagent for measuring ion calcium in blood samples as described in the first aspect. The chip body is provided with a colorimetric hole and a diluent tank. The freeze-dried reagent ball is accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank.

[0021] The third aspect of the present invention is to provide a method for measuring ion calcium in a blood sample, the method being applied to the biochemical analysis chip as described in the second aspect, the method comprising: S1, obtaining a whole blood sample, sending the whole blood sample into the centrifugal structure of the biochemical analysis chip for centrifugal treatment, and then sending the whole blood sample into the quantitative structure to separate quantitative plasma; S2, diluting the quantitative plasma with the diluent to form a diluted plasma sample; S3, filling the diluted plasma sample into the colorimetric well, so that the diluted plasma sample fully reacts with the freeze-dried reagent ball in the colorimetric well containing the blood sample ion calcium determination reagent; wherein, during the reaction, the temperature of the biochemical analysis chip is heated to an appropriately selected temperature; S4, testing the absorbance change of the solution in the colorimetric well at a specific wavelength; S5, based on the absorbance change, obtaining the ion calcium concentration value of the whole blood sample through a data analysis system.

[0022] Preferably, S4 includes: using a spectrophotometer or an automatic analytical instrument commonly used in clinical practice to test the increase rate of the absorbance value of the solution in the colorimetric well; the specific wavelength of the light source generated by the spectrophotometer is 500-700 nm.

[0023] Beneficial effects of the reagent, biochemical analysis chip and assay method of the present invention: (1) The present invention provides a freeze-dried reagent ball and a diluent for measuring blood sample ion calcium, wherein the freeze-dried reagent ball is made by freeze-drying a droplet of a blood sample ion calcium measuring reagent. Compared with the conventional enzymatic analysis, the present invention improves the reagent composition, adds a surfactant to the diluent to increase the solubility of phospholipids and the repeatability of the reagent reaction time. The configuration process and formula of the measuring reagent are innovated to improve the stability of the reagent, and the freeze-drying process is used to further improve the stability and repeatability of the reagent.

[0024] (2) The present invention also provides a biochemical analysis chip, which includes a chip body, a freeze-dried reagent ball and a diluent. The chip body is provided with a colorimetric hole and a diluent tank. The freeze-dried reagent ball is accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank of the chip body. The chip contains a sample quantitative separation structure, a mixing mechanism, a reaction hole and a hole modification. The chip is the core part of the blood sample ion calcium determination. The blood sample pretreatment, quantitative separation of plasma, quantitative mixing of plasma and diluent, and the final ion calcium quantitative detection reaction are all completed in the chip.

[0025] (3) The present invention also provides a method for measuring ionized calcium in blood samples, which is applied to a biochemical analysis chip, and the overall detection process of the reagent, chip and instrument is a complete solution for rapid quantitative detection of ionized calcium in blood samples. The method includes: a biochemical analysis chip is combined with an appropriate detection method to complete the quantitative detection of ionized calcium, specifically including: controlling the reaction time, reaction temperature, measurement of the absorbance change rate, and calculation of the ionized calcium concentration. The method can achieve instant detection of blood sample calcium ions, and only requires a small amount of whole blood sample to be dripped into the sample hole of the chip, and the blood sample ionized calcium concentration value can be obtained after about 1-10 minutes.

[0026] (4) In summary, the present invention aims to solve the problems of complicated detection steps, complex operation and long time consumption in the prior art, and proposes a freeze-dried reagent ball, a biochemical analysis chip and a method for measuring calcium ions in blood samples. Through simple operation, the calcium ion level in body fluids can be quantitatively measured with high precision. At the same time, this product has established a system for micro-determination of calcium ions in blood samples, and the purpose of accurate determination can be achieved using a small amount of whole blood samples. This method saves costs, is easy to operate, has simple equipment, requires less blood, has good reagent stability and specificity, and is easy to be promoted and used clinically. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the related technologies, the drawings required for use in the specific embodiments or the related technical descriptions will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 The figure is a flow chart of a method for measuring ionized calcium in a blood sample according to an embodiment of the present invention.

[0029] Figure 2 , Figure 3 and Figure 4The first schematic diagram, the second schematic diagram and the third schematic diagram respectively illustrate the ability of three biochemical analysis chips provided according to embodiments of the present invention to measure ionized calcium.

[0030] In the first, second and third schematic diagrams, the ordinate is the change in absorbance at 530 nm per minute, and the abscissa is the calcium ion concentration. The fitting expressions of the three biochemical analysis chips and the values ​​of the correlation coefficient R² are shown in Figure 2 , Figure 3 and Figure 4 The expression and value in . Goodness of Fit refers to the degree of fit of the regression line to the observed values. The statistic that measures the goodness of fit is the coefficient of determination (also known as the coefficient of determination or correlation coefficient) R². The maximum value of R² is 1. The closer the value of R² is to 1, the better the fit of the regression line to the observed values; conversely, the smaller the value of R², the worse the fit of the regression line to the observed values.

[0031] Figure 5 The values ​​measured by the ion selective electrode method are taken as the ordinate, and the ion calcium concentration values ​​measured by the biochemical analysis chip provided by the embodiment of the present invention are taken as the abscissa. The ion calcium concentration (mg / dl) tested by the ion selective electrode represents the results of the clinical correlation analysis of the biochemical analysis chip provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0034] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0035] The term "about" is used herein in conjunction with numerical values ​​to include the normal variation in measurements expected by those skilled in the art and should be understood to have the same meaning as "approximately" and to cover the typical margin of error, eg, ±5% of the stated value.

[0036] Terms such as "a," "an," and "the" are not intended to refer to only a singular entity, but include the general class that may be illustrated using a specific example.

[0037] Unless otherwise indicated, any amount (eg, concentration) of a component in a composition given as a percentage (%) refers to the weight percent per volume.

[0038] As used herein, the term "lyophilization" (also known as "freeze drying," "freeze drying," or "freeze drying") refers to a low-temperature dehydration process that involves freezing the product and reducing the pressure to remove the ice by sublimation.

[0039] As used herein, the term "lyoprotectant" refers to stabilization during the entire freeze-drying process. Freeze-drying of biological materials such as proteins, peptides, and biopharmaceuticals often requires such stabilization. This is because complex biological molecules often require an appropriate amount of residual water to maintain structure and function. Therefore, "lyoprotectants" protect the structure and / or function of biologically active ingredients during freeze-drying (e.g., prevent aggregation, improve bioavailability, and increase stability).

[0040] An embodiment of the present invention provides a reagent for measuring blood sample ion calcium, comprising: a blood sample ion calcium measuring reagent and a diluent, wherein the blood sample ion calcium measuring reagent comprises the following components: phospholipase D [EC3.1.4.4], choline oxidase [1.1.3.17], peroxidase [EC1.11.1.7], a first color developer, a divalent metal salt, a first buffer, a first additive and a cryoprotectant; the diluent comprises the following components: phospholipids, a second color developer, a surfactant, a second additive and a second buffer; the blood sample ion calcium measuring reagent exists in the form of a freeze-dried reagent ball, and the freeze-dried reagent ball is made by freeze-drying droplets of the blood sample ion calcium measuring reagent.

[0041] As a preferred embodiment, the source of the phospholipase D [EC3.1.4.4] is not limited; for example, animal (e.g., pig pancreas, rat brain) or plant source (e.g., cabbage, carrot, peanut, spinach, cotton) or microbial source (e.g., Streptomyces), including phospholipase D prepared by integrating the gene of a microorganism into a microorganism such as Escherichia coli by gene recombination technology, and phospholipase D prepared by a microorganism with improved characteristics by genetic modification. From the perspective of stability, availability and batch difference, phospholipase D of microbial origin is preferred. In the present invention, phospholipase D derived from a microorganism belonging to the genus Streptomyces is preferably used.

[0042] As a preferred embodiment, the concentration of the phospholipase D [EC3.1.4.4] is 0.0001-10 U / ml, and a more preferred concentration is 0.0005-5 U / ml.

[0043] As a preferred embodiment, the source of the choline oxidase [1.1.3.17] is not limited. For example, choline oxidase from microorganisms, Arthrobacter sphaeroides or Alcaligenes can be used.

[0044] As a preferred embodiment, the concentration of the choline oxidase is 0.1-10 U / ml.

[0045] As a preferred embodiment, the peroxidase [EC1.11.1.7] includes peroxidase obtained from horseradish.

[0046] As a preferred embodiment, the concentration of the peroxidase [EC1.11.1.7] is 0.1-300 U / ml; more preferably, the concentration is 0.5-200 U / ml.

[0047] As a preferred embodiment, the first color developer includes one or more of 4-aminoantipyrine (4-AA) and 3-methylbenzothiazolinonehydrazone (MBTH).

[0048] As a preferred embodiment, the concentration of 4-aminoantipyrine (4-AA) is 0.001-10 wt %; more preferably, the concentration is 0.01-5 wt %.

[0049] As a preferred embodiment, the divalent metal salt includes one or more of a divalent alkaline earth metal salt (such as a barium salt) and a divalent transition metal salt (such as a zinc salt, a manganese salt, etc.). Suitable divalent metal salts include chlorides or sulfates soluble in aqueous solution.

[0050] As a preferred embodiment, the concentration of the divalent metal salt is 0.1-200 mmol / L; a more preferred concentration is 0.5-100 mmol / L.

[0051] As a preferred embodiment, the first buffer includes a buffer capable of buffering to a pH value in the range of 5 to 9; including: one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 2-morpholineethanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(trihydroxymethyl)methane.

[0052] As a preferred embodiment, the concentration of the first buffer is 1-100 mmol / L to adjust the pH value to the range of 4.0-10.0; a more preferred concentration is 10-500 mmol / L to adjust the pH value to the range of 6.0-9.0.

[0053] As a preferred embodiment, the first additive comprises a monovalent metal salt, a sugar, a sugar alcohol or a protein, and also comprises a chelating agent, an amine, a basic amino acid or a heterocyclic compound; the monovalent metal salt comprises chloride, sulfate or acetate of sodium or potassium; the sugar comprises sucrose or lactose; the sugar alcohol comprises sorbitol or mannitol; the protein comprises bovine serum albumin.

[0054] As a preferred embodiment, the concentration of the first additive is 0.1-30wt%.

[0055] As a preferred embodiment, the cryoprotectant acts as a stabilizer and an excipient at the same time, and the cryoprotectant includes sugars, polyols, amino acids, proteins, peptide excipients, and cyclic oligosaccharide-based polymers; wherein, The sugars include one or more of glucose, sucrose, lactose, maltose and trehalose; The polyols include one or more of mannitol, inositol, glycerol, ethylene glycol and polyethylene glycol; The amino acids include trimethylglycine, glycine, arginine or any salt thereof; The protein and peptide excipients include one or more of mucopolysaccharide protein, casein and bovine serum albumin; The cyclic oligosaccharide-based polymer includes α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether, or any derivatives thereof, and any combination thereof.

[0056] In addition to being used as stabilizers, mannitol and sucrose are also used as excipients, which help to give the freeze-dried reagent balls a good appearance, making the freeze-dried reagent balls loose and porous and easy to reconstitute.

[0057] As a preferred embodiment, the concentration of the cryoprotectant is 0.1-60 wt %.

[0058] As a preferred embodiment, the phospholipids are not particularly limited as long as they contain choline as their basic part. Examples of available phospholipids are lysolecithin obtained from egg yolk, beef liver, etc., such as L-α-lysophosphatidylcholine, L-α-lysophosphatidylpalmitoylcholine and L-α-lysophosphatidylstearoylcholine, lecithin obtained from egg yolk, soybean, among which lysolecithin derived from egg yolk is preferred because of its availability, excellent water solubility and stable quality.

[0059] As a preferred embodiment, the concentration of the phospholipid is 0.2-100 mg / ml; a more preferred concentration is 0.5-50 mg / ml.

[0060] As a preferred embodiment, the second developer includes 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA), sodium 3,5-dichloro-2-hydroxybenzenesulfonate (DHBS), phenol, toluidine, aniline and derivatives of these compounds; aniline derivatives include N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt (HDAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt (TOOS), N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt (TOPS), N-ethyl-N -(2-carboxy-3-sulfopropyl)-3-methoxyaniline sodium salt (ADOS), N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt (ADPS), N-ethyl-N-(3-sulfopropyl)aniline sodium salt (ALPS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3'5-dimethoxyaniline sodium salt (DAOS), N'N-di(4-sulfobutyl)-3,5-dimethylaniline disodium salt (MADB) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt (MAOS). The second color developer is most preferably: 2,4,6-tribromo-3-hydroxybenzoic acid (TBHBA) or 3,5-dichloro-2-hydroxybenzenesulfonate sodium (DHBS).

[0061] As a preferred embodiment, the concentration of the second developer is 0.001-10wt%; more preferably, the concentration is 0.01-5wt%.

[0062] As a preferred embodiment, the surfactant includes a nonionic surfactant and an anionic surfactant; the nonionic surfactant includes polyethylene glycol monooctylphenyl ether (TritonX-100), sorbitol monolaurate (Span20), sorbitan monopalmitate (Span40), polyoxyethylene lauryl ether (Brij35), polyoxyethylene cetyl ester (Brij58), polyoxyethylene sorbitol monolaurate (Brij58), polyoxyethylene sorbitol monolaurate (Span40), Tween 20, Tween 40, Tween 80 and Tween 60; the anionic surfactant includes sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate and sodium lauryl sarcosine. These surfactants can be used alone or in combination of two or more.

[0063] In this embodiment, the most preferred combinations of the surfactants are: a combination of TritonX-100, sodium dodecyl sulfate and sodium lauryl benzene sulfonate; and a combination of sodium deoxycholate, TritonX-100 and sodium dodecyl benzene sulfonate.

[0064] As a preferred embodiment, the concentration of the surfactant is 0.001-10 wt %; more preferably, the concentration is 0.01-1 wt %.

[0065] As a preferred embodiment, the second buffer includes a buffer capable of buffering to a pH value in the range of 5 to 8; including: one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-di(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 2-morpholineethanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(trihydroxymethyl)methane.

[0066] As a preferred embodiment, the concentration of the second buffer is 1-1000 mmol / L to adjust the pH value to the range of 4.0-10.0; a more preferred concentration is 10-100 mmol / L to adjust the pH value to the range of 5.0-8.0.

[0067] In this embodiment: The reagent contains 0.0001-10U / ml, preferably 0.0005-5U / ml of phospholipase D; 0.1-50U / ml, preferably 0.1-10U / ml of choline oxidase; 0.1-300U / ml, preferably 0.5-200U / ml of peroxidase; 0.001-10wt%, preferably 0.01-5wt% of 4-aminoantipyrine; 0.1-200mmol / L, preferably 0.5-100mmol / L of divalent metal salt; 0.1-60wt% of excipient; 0.1-30wt% of additive; 1-100mmol / L of buffer to adjust pH to 4.0-10.0. It is particularly preferred that the buffer concentration is 10-500mmol / L to adjust pH to the range of 6.0-9.0.

[0068] The diluent contains 0.2 to 100 mg / ml, preferably 0.5 to 50 mg / ml of lysolecithin; 0.001 to 10 wt%, preferably 0.01 to 5 wt% of 2,4,6-tribromo-3-hydroxybenzoic acid; 0.001 to 10 wt%, preferably 0.01 to 1 wt% of Triton X-100, and a buffer is added at a concentration of 1 to 1000 mmol / L to adjust the pH value to a range of 4.0 to 10.0. Particularly preferably, the concentration is adjusted to 10-100 mmol / L to adjust the pH value to a range of 5.0-8.0.

[0069] An embodiment of the present invention also provides a biochemical analysis chip for measuring ion calcium in a blood sample. The biochemical analysis chip includes a chip body and a reagent for measuring ion calcium in a blood sample as described in the first aspect. The chip body is provided with a colorimetric hole and a diluent tank. The freeze-dried reagent ball is accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank.

[0070] An embodiment of the present invention further provides a method for measuring ion calcium in a blood sample, the method being applied to the biochemical analysis chip as described in the second aspect, the method comprising: S1, obtaining a whole blood sample, sending the whole blood sample into the centrifugal structure of the biochemical analysis chip for centrifugal treatment, and then sending the whole blood sample into the quantitative structure to separate quantitative plasma; S2, diluting the quantitative plasma with the diluent to form a diluted plasma sample; S3, filling the diluted plasma sample into the colorimetric well, so that the diluted plasma sample and the freeze-dried reagent ball in the colorimetric well containing the blood sample ion calcium determination reagent are fully reacted; wherein, during the reaction, the temperature of the biochemical analysis chip is heated to an appropriately selected temperature; in this embodiment, the appropriately selected temperature is a value between 25°C and 37°C; and the reaction time is 1 to 30 minutes; S4, testing the absorbance change of the solution in the colorimetric well at a specific wavelength; In this embodiment, a spectrophotometer or an automatic analytical instrument commonly used in clinical practice is used to test the rate of increase of the absorbance value of the solution in the colorimetric well; the specific wavelength of the light source generated by the spectrophotometer is 500-700nm; most preferably, the specific wavelength is one of 530nm, 546nm, 583nm, 605nm, 630nm, 670nm or 700nm.

[0071] S5, based on the absorbance change, obtaining the ion calcium concentration value of the whole blood sample through a data analysis system.

[0072] The embodiment of the present invention provides a freeze-dried reagent ball for measuring ion calcium in human blood samples, wherein the freeze-dried reagent ball is made by freeze-drying droplets of a blood sample ion calcium measuring reagent. Specifically, the blood sample ion calcium measuring reagent includes the following components: phospholipase D, choline oxidase, peroxidase, a color developer, a divalent metal salt, a buffer, an additive, and an excipient.

[0073] In some embodiments, the preparation method of the blood sample ion calcium determination reagent is as follows: (1) first prepare an enzyme protection solution: comprising a certain concentration of divalent metal salt, buffer, additives, and excipients; (2) prepare an enzyme solution: comprising a certain concentration of phospholipase D, choline oxidase, peroxidase, buffer, and additives; (3) prepare a color development solution: comprising a certain concentration of buffer, color developer, and additives; (4) color developer protection solution: comprising a certain concentration of buffer, additives, and excipients. (1) and (2) are fully mixed in a certain proportion and order, and (3) and (4) are fully mixed. Finally, the two groups of mixed solutions are mixed in a certain proportion and fixed volume to form a blood sample ion calcium determination reagent.

[0074] Freeze drying traditionally consists of two major steps: (1) freezing of the solution, and (2) drying of the frozen solid under vacuum. The drying step is further divided into two stages: primary drying and secondary drying. The primary drying step attempts to remove the frozen water or solvent (sublimation), and the secondary drying step attempts to remove the unfrozen "bound" water or solvent from the solidified solution (desorption).

[0075] In some embodiments, the method for preparing the lyophilized reagent pellets comprises the following steps: (1) Freezing of the solution: Drops of the blood sample ion calcium determination reagent are dropped into liquid nitrogen using a precise quantitative dispensing system, so that the droplets of the blood sample ion calcium determination reagent form ice balls; for example, droplets of the blood sample ion calcium determination reagent can be dropped into liquid nitrogen using a dispensing machine, so that the droplets of the blood sample ion calcium determination reagent condense into ice balls in the liquid nitrogen. Those skilled in the art can adjust the size of the droplets of the mixed solution dropped into the liquid nitrogen according to actual needs, and adjust the volume of the ice ball by controlling the size of the droplets. Optionally, in some embodiments, the volume of the blood sample ion calcium determination reagent is 1-5 μl.

[0076] (2) Drying of frozen solids under vacuum: freeze-drying the ice balls to obtain freeze-dried reagent balls; In this embodiment, the ice balls are placed in a vacuum freeze dryer for freeze drying to form freeze-dried reagent balls, and after nitrogen re-pressurization, the freeze-dried reagent balls are collected and stored in a dry aluminum bottle.

[0077] Freeze drying is the process of removing water or other solvents by freeze drying, which stabilizes the reagent by greatly reducing the degradation rate of the components. The removal of the solvent is first accomplished by freezing the solution, so that the freezing process separates one or more solvents from the solute and fixes any unfrozen solvent molecules in the interstitial areas between the frozen solvent crystals. The solvent is then removed by sublimation (primary drying) and then by desorption (secondary drying). After freeze drying, the phospholipase D, choline oxidase, peroxidase, colorimetric agent, divalent metal salt, buffer, additive, and cryoprotectant in the blood sample ion calcium assay reagent remain in the ice shelf during freezing, so the freeze-dried reagent ball after freeze drying is loose and porous, and its volume remains basically the same as before freeze drying. Since the freeze-dried reagent ball is always in a frozen state before drying, the ice crystals are evenly distributed in the components of the blood sample ion calcium assay reagent. During the sublimation process, the components will not be concentrated due to dehydration. Therefore, the freeze-dried reagent ball after freeze drying is spongy, loose and porous, and is very easy to dissolve in water and return to its original state. The freeze-dried reagent balls provided by the embodiment of the present invention are suitable for large-scale batch production, and the solid freeze-dried reagent balls are more convenient to store and transport.

[0078] The embodiment of the present invention provides a freeze-dried reagent ball and a diluent for determining the content of ionized calcium in a blood sample, and the determination principle is as follows: lecithin produces choline under the action of phospholipase, choline produces hydrogen peroxide under the action of choline oxidase, and hydrogen peroxide produces a colored product under the action of peroxidase and a dye, so that the reaction solution has an absorption peak at a certain wavelength. Because the expression of the activity of phospholipase D in the system essentially requires the presence of calcium ions, and the activity of phospholipase D increases proportionally with the concentration of calcium ions, the absorbance value of the reaction solution at this wavelength is proportional to the concentration of calcium ions within a certain range. Therefore, the concentration of calcium ions in the serum sample can be calculated by the rate of increase of absorbance.

[0079] In the reaction system, by containing or coexisting at least one substance selected from the group consisting of divalent metal ions, chelating agents, amines, basic amino acids or heterocyclic compounds, the measurement range of calcium concentration in the sample can be expanded to a high concentration range.

[0080] In addition, by including or coexisting a nonionic surfactant in the assay reagent or in the assay reaction, the reaction process when measuring the calcium concentration in the sample can be stabilized. By including or coexisting anionic surfactant in the assay reagent or in the assay reaction, the sensitivity of the calcium concentration measurement can be increased, and even trace concentrations of ionized calcium can be measured.

[0081] The embodiment of the present invention also provides a biochemical analysis chip, which includes a chip body and the above-mentioned freeze-dried reagent balls and diluent. The chip body is provided with a colorimetric hole and a diluent tank, the freeze-dried reagent balls are accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank.

[0082] Figure 1 A flow chart of a method for measuring ionized calcium in a blood sample provided by an embodiment of the present invention is shown in FIG. Figure 1 The method shown comprises the following steps: S1, obtaining a whole blood sample, sending the whole blood sample into the centrifugal structure of the biochemical analysis chip for centrifugal treatment, and then sending the whole blood sample into the quantitative structure to separate quantitative plasma; S2, diluting the quantitative plasma with the diluent to form a diluted plasma sample; S3, filling the diluted plasma sample into the colorimetric well, so that the diluted plasma sample fully reacts with the freeze-dried reagent ball in the colorimetric well containing the blood sample ion calcium determination reagent; wherein, during the reaction, the temperature of the biochemical analysis chip is heated to an appropriately selected temperature; S4, testing the absorbance change of the solution in the colorimetric well at a specific wavelength; S5, based on the absorbance change, obtaining the ion calcium concentration value of the whole blood sample through a data analysis system.

[0083] In the centrifugal microfluidic biochemical analysis system, due to the inherent centrifugal properties, the sample can be detected without centrifugation, so serum, plasma and whole blood can be used for detection. In some embodiments, the biochemical analysis chip includes a sample well, a sample slot, a diluent slot, a sample quantitative slot, a diluent quantitative slot and a mixing slot. When the biochemical analysis chip is used for testing, the sample is added to the sample slot from the sample well by a pipette. The diluent is made to enter the diluent quantitative slot from the diluent slot by centrifugation, the serum sample is made to enter the sample quantitative slot from the sample slot, and the diluent in the diluent quantitative slot and the serum sample in the sample quantitative slot enter the mixing slot to dilute the serum sample by the diluent. The serum sample diluted in the mixing slot is then filled into the colorimetric well by centrifugation, and after the serum sample to be diluted fully reacts with the freeze-dried reagent ball in the colorimetric well, the absorbance change of the solution in the colorimetric well is tested by a spectrophotometer.

[0084] The wavelength of the light source generated by the spectrophotometer is 500-700nm. In order to improve the test accuracy and broaden the test range, the wavelength of the light source can be one of 546nm, 605nm, 630nm, 670nm or 700nm.

[0085] Figure 2 , Figure 3 and Figure 4 A first schematic diagram, a second schematic diagram and a third schematic diagram respectively show the ability of three biochemical analysis chips provided in embodiments of the present invention to measure ionized calcium; In the first, second and third schematic diagrams, the ordinate is the change in absorbance at 530 nm per minute, and the abscissa is the calcium ion concentration. The fitting expressions of the three biochemical analysis chips and the values ​​of the correlation coefficient R² are shown in Figure 2 , Figure 3 and Figure 4 The expression and value in . Goodness of Fit refers to the degree of fit of the regression line to the observed values. The statistic that measures the goodness of fit is the coefficient of determination (also known as the coefficient of determination) R². The maximum value of R² is 1. The closer the value of R² is to 1, the better the fit of the regression line to the observed values; conversely, the smaller the value of R², the worse the fit of the regression line to the observed values.

[0086] To further illustrate the technical solution of the present invention, several embodiments of the method for preparing the blood sample ion calcium determination reagent of the present invention are provided below. Example 1

[0087] The blood sample ion calcium determination reagent of this embodiment includes the following components as shown in Table 1 and Table 2: An assay reagent and diluent having the following composition were prepared.

[0088] Table 1

[0089] In this embodiment, the freeze-dried reagent ball is prepared by the preparation method of the freeze-dried reagent ball, and the volume of the blood sample ion calcium determination reagent droplet is 1.5ul.

[0090] Table 2

[0091] The biochemical analysis chip in this embodiment includes a chip body and the freeze-dried reagent balls and diluent provided in this embodiment. The above blood sample ion calcium determination method is used to detect the absorbance change per minute of the serum sample, and the wavelength of the light source selected during the test is 530nm.

[0092] The biochemical analysis chip of the present invention is used to test the ion calcium concentration, and the test results are as follows: Figure 2 The relationship between calcium ion concentration and absorbance change rate is shown in Figure 2 As shown, in the range of ionized calcium concentration of 0.5-16 mg / dl, the change in absorbance per minute is linearly related to the ionized calcium concentration. Example 2

[0093] To demonstrate the applicability of zinc chloride as a divalent metal salt and different additives, calcium ion level determination was performed in the same manner as in Example 1 except that the following reagents were used.

[0094] The differences between this embodiment and embodiment 1 are shown in Table 3 and Table 4: Table 3

[0095] Table 4

[0096] The wavelength of the light source generated by the spectrophotometer used in the test is 530nm, and the results obtained are as follows Figure 3 The relationship between calcium ion concentration and absorbance change rate is shown in Figure 3 As shown, in the range of ionized calcium concentration of 0.5-16 mg / dl, the change in absorbance per minute is linearly related to the ionized calcium concentration.

[0097] Example 3 To demonstrate the applicability of lecithin as a substrate, calcium ion level assay was performed in the same manner as in Example 1 except that the following reagent solutions were used.

[0098] The differences between this embodiment and Embodiment 1 and Embodiment 2 are shown in Table 5 and Table 6: Table 5

[0099] Table 6

[0100] The results obtained are as follows Figure 4 As shown, there is a linear relationship between ion calcium concentration and the rate of change of absorbance at 530 nm within a certain concentration range.

[0101] Example 4 Specificity test: A calcium ion solution of 0, 5 or 10 mg / dl was prepared, and each concentration was divided into 8 tubes, labeled as sample 1 to sample 8, sample 1 contained 29 mg / dl magnesium ion, sample 2 contained 1.1 mg / dl manganese ion, sample 3 contained 1.3 mg / dl copper ion, sample 4 contained 1.3 mg / dl zinc ion, sample 5 contained 1.1 mg / dl iron ion, sample 6 contained 30 mg / dl bilirubin, sample 7 contained 500 mg / dl hemoglobin, and sample 8 contained 50 mg / dl ascorbic acid. Next, the reagents of the following compositions were prepared as shown in Tables 7 and 8.

[0102] Table 7

[0103] Table 8

[0104] The spectrophotometer used in the test generates a light source with a wavelength of 530 nm, and the process of Example 1 is repeated to measure the change in absorbance.

[0105] The test results are shown in Table 9 below: Table 9

[0106] It can be seen from Table 9 above that although different concentrations of magnesium ions, manganese ions, copper ions, zinc ions, iron ions, ascorbic acid, bilirubin or hemoglobin were added to the samples, the presence of the above interfering substances had very little effect on the test results of the biochemical analysis chip proposed in the present invention, which shows that the biochemical analysis chip and the test method proposed in the present invention have good specificity.

[0107] Example 5 Precision Test: The biochemical analysis chip and the test method provided in Example 4 of the present invention were used to perform sample testing. A solution containing 10 mg / dl of calcium ions was used as a standard, and serum samples with known calcium ion concentrations of 5 mg / dl and 10 mg / dl were tested 31 times each.

[0108] According to formulas (1)-(3), serum samples with calcium ion concentrations of 5 mg / dl and 10 mg / dl were tested 31 times each, and the average value of the measured values ​​was ( ) were 5.26mg / dl and 9.88mg / dl, the standard deviation (SD) was 0.075mg / dl and 0.112mg / dl, and the coefficient of variation (CV) was 1.43% and 1.13%, respectively. This indicates that the biochemical analysis chip and the test method provided by the present invention have sufficient precision.

[0109] (1); (2); (3); Where Xi is the concentration value measured for the i-th time, and n is the number of tests.

[0110] Example 6 Clinical correlation analysis: The biochemical analysis chip and the test method provided in Example 4 of the present invention were used to perform sample testing. A 10 mg / dl calcium ion solution was used as a calibrant to measure the ion calcium content of 25 serum samples. In addition, the ion selective electrode method was used as a reference method to simultaneously measure the serum ion calcium content of the same 25 serum samples. The corresponding measured serum ion calcium concentration value (in mg / dl) is as follows: Figure 5 As shown, Figure 5 In the figure, the ordinate value is the serum ion calcium concentration in the serum sample measured by the ion selective electrode, and the abscissa value is the serum ion calcium concentration in the serum sample measured by the biochemical analysis chip provided in Example 4 of the present invention.

[0111] Figure 5 The values ​​measured by the ion selective electrode method are taken as the ordinate, and the ion calcium concentration values ​​measured by the biochemical analysis chip provided by the embodiment of the present invention are taken as the abscissa. The ion calcium concentration (mg / dl) tested by the ion selective electrode represents the results of the clinical correlation analysis of the biochemical analysis chip provided by the embodiment of the present invention.

[0112] The linear regression equation between the two groups of test results of the biochemical analysis chip and the ion selective electrode provided by the embodiment of the present invention is shown in the following equation (4): y=0.8817x+0.8903(4); Correlation coefficient R 2 =0.9809, the closer the correlation coefficient is to 1, the stronger the correlation between the two sets of data. Therefore, the biochemical analysis chip provided by the embodiment of the present invention has a strong correlation with the test results of the ion selective electrode.

[0113] Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiments can be implemented by software, or by means of software plus necessary general hardware platforms. Based on such understanding, the technical solutions of the above embodiments can be embodied in the form of software products, which can be stored in a non-volatile storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.), including several instructions for enabling a computer device (which can be a personal computer, a server, or a network device, etc.) to execute the methods described in the various embodiments of the present invention.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A reagent for measuring ionized calcium in a blood sample, characterized in that: include: A blood sample ion calcium assay reagent and a diluent, wherein the blood sample ion calcium assay reagent comprises the following components: phospholipase D [EC3.1.4.4], choline oxidase [1.1.3.17], peroxidase [EC1.11.1.7], a first color developer, a divalent metal salt, a first buffer, a first additive and a cryoprotectant; the diluent comprises the following components: phospholipids, a second color developer, a surfactant, a second additive and a second buffer; the blood sample ion calcium assay reagent exists in the form of a freeze-dried reagent ball, and the freeze-dried reagent ball is made by freeze-drying droplets of the blood sample ion calcium assay reagent.

2. A reagent for determining ionized calcium in a blood sample according to claim 1, characterized in that: The sources of the phospholipase D [EC3.1.4.4] include: animal source, plant source or microbial source; the phospholipase D [EC3.1.4.4] includes: phospholipase D prepared by integrating microbial genes into microorganisms through gene recombination technology and phospholipase D prepared by microorganisms with improved characteristics through genetic modification; the concentration of the phospholipase D [EC3.1.4.4] is 0.0001-10U / ml.

3. A reagent for determining ionized calcium in a blood sample according to claim 1, characterized in that: The choline oxidase [1.1.3.17] includes: choline oxidase of microbial origin; the microbial source includes spherical Arthrobacter or Alcaligenes; the concentration of the choline oxidase is 0.1-10U / ml.

4. A reagent for determining ionized calcium in a blood sample according to claim 1, characterized in that: The peroxidase [EC1.11.1.7] includes peroxidase obtained from horseradish; the concentration of the peroxidase [EC1.11.1.7] is 0.1-300 U / ml.

5. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The first developer includes one or more of 4-aminoantipyrine (4-AA) and 3-methylbenzothiazolinone hydrazone (MBTH); the concentration of the first developer is 0.001-10 wt %.

6. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The divalent metal salt includes one or more of a divalent alkaline earth metal salt or a divalent transition metal salt; the divalent alkaline earth metal salt or the divalent transition metal salt includes a chloride or a sulfate soluble in an aqueous solution; the concentration of the divalent metal salt is 0.1-200 mmol / L.

7. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The first buffer includes a buffer capable of buffering to a pH value in the range of 5 to 9; including: one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 2-morpholineethanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane); the concentration of the first buffer is 1-100 mmol / L to adjust the pH value to the range of 4.0 to 10.0 or the concentration of the first buffer is 10-500 mmol / L to adjust the pH value to the range of 6.0 to 9.

0.

8. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The first additive includes a monovalent metal salt, sugar, sugar alcohol or protein, and also includes a chelating agent, amines, basic amino acids or heterocyclic compounds; the monovalent metal salt includes chloride, sulfate or acetate of sodium or potassium; the sugar includes sucrose or lactose; the sugar alcohol includes sorbitol or mannitol; the protein includes bovine serum albumin; the concentration of the first additive is 0.1-30wt%.

9. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The cryoprotectant acts as a stabilizer and an excipient at the same time, and includes sugars, polyols, amino acids, proteins, peptide excipients, and cyclic oligosaccharide-based polymers; wherein, The sugars include one or more of glucose, sucrose, lactose, maltose and trehalose; The polyols include one or more of mannitol, inositol, glycerol, ethylene glycol and polyethylene glycol; The amino acids include trimethylglycine, glycine, arginine or any salt thereof; The protein and peptide excipients include one or more of mucopolysaccharide protein, casein and bovine serum albumin; The cyclic oligosaccharide-based polymer comprises α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, β-cyclodextrin sulfobutyl ether, or any derivatives thereof, and any combination thereof; the concentration of the cryoprotectant is 0.1-60 wt %.

10. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The phospholipids include lysophosphatidylcholine or phosphatidylcholine; the concentration of the phospholipids is 0.2-100 mg / ml.

11. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The second color developer includes 2,4,6-tribromo-3-hydroxybenzoic acid, sodium 3,5-dichloro-2-hydroxybenzenesulfonate, phenol, toluidine, aniline and derivatives of these compounds; aniline derivatives include N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline sodium salt, N-ethyl-N-(3-sulfopropyl)-3-methylaniline sodium salt, N-ethyl-N-(2-carboxy-3-sulfopropyl)-3-methylaniline sodium salt, The invention relates to one of oxyaniline sodium salt, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline sodium salt, N-ethyl-N-(3-sulfopropyl)aniline sodium salt, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3'5-dimethoxyaniline sodium salt, N'N-di(4-sulfobutyl)-3,5-dimethylaniline disodium salt (MADB) and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline sodium salt; the concentration of the second developer is 0.001-10wt%.

12. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The surfactant includes a nonionic surfactant and an anionic surfactant; the nonionic surfactant includes polyethylene glycol monooctylphenyl ether, sorbitan monolaurate, sorbitan monopalmitate, polyoxyethylene lauryl ether, polyoxyethylene cetyl ester, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monolaurate (Span40), Tween 20, Tween 40, Tween 80 and Tween 60; the anionic surfactant includes sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium cholate, sodium deoxycholate and sodium lauryl sarcosinate; the nonionic surfactant and the anionic surfactant are used alone or in combination of two or more; the concentration of the surfactant is 0.001-10wt%.

13. A reagent for measuring ionized calcium in a blood sample according to claim 1, characterized in that: The second buffer is a buffer capable of buffering to a pH value in the range of 5 to 8; including: one or more of tris(hydroxymethyl)aminomethane, tris(hydroxymethyl)methylglycine, 4-hydroxyethylpiperazinepropanesulfonic acid, 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethaneethanesulfonic acid, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, 3-morpholinepropanesulfonic acid, 2-morpholineethanesulfonic acid, 1,4-piperazinediethanesulfonic acid and bis(2-hydroxyethyl)amino(tris(hydroxymethyl)methane); the concentration of the second buffer is 1 to 1000 mmol / L to adjust the pH value to 4.0-10 or the concentration of the second buffer is 10 to 100 mmol / L to adjust the pH value to 5.0-8.

0.

14. A biochemical analysis chip, characterized in that: Used for the determination of blood sample ion calcium, the biochemical analysis chip comprises a chip body and a reagent for determining blood sample ion calcium as described in any one of claims 1-13, the chip body is provided with a colorimetric hole and a diluent tank, the freeze-dried reagent ball is accommodated in the colorimetric hole, and the diluent is accommodated in the diluent tank.

15. A method for measuring ionized calcium in a blood sample, the method being applied to the biochemical analysis chip according to claim 14, characterized in that The determination method comprises: S1, obtaining a whole blood sample, sending the whole blood sample into the centrifugal structure of the biochemical analysis chip for centrifugal treatment, and then sending the whole blood sample into the quantitative structure to separate quantitative plasma; S2, diluting the quantitative plasma with the diluent to form a diluted plasma sample; S3, filling the diluted plasma sample into the colorimetric well, so that the diluted plasma sample fully reacts with the freeze-dried reagent ball in the colorimetric well containing the blood sample ion calcium determination reagent; wherein, during the reaction, the temperature of the biochemical analysis chip is heated to an appropriately selected temperature; S4, testing the absorbance change of the solution in the colorimetric well at a specific wavelength; S5, based on the absorbance change, obtaining the ion calcium concentration value of the whole blood sample through a data analysis system.

16. The measuring method according to claim 15, characterized in that The step S4 includes: using a spectrophotometer or an automatic analytical instrument commonly used in clinical practice to test the increase rate of the absorbance value of the solution in the colorimetric well; the specific wavelength of the light source generated by the spectrophotometer is 500-700 nm.

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