CN111948153A - Freeze-drying reagent ball, biochemical analysis chip and serum total calcium determination method - Google Patents
Freeze-drying reagent ball, biochemical analysis chip and serum total calcium determination method Download PDFInfo
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- CN111948153A CN111948153A CN202010685144.0A CN202010685144A CN111948153A CN 111948153 A CN111948153 A CN 111948153A CN 202010685144 A CN202010685144 A CN 202010685144A CN 111948153 A CN111948153 A CN 111948153A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/314—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths
- G01N2021/3148—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry with comparison of measurements at specific and non-specific wavelengths using three or more wavelengths
Abstract
The invention relates to the technical field of serum total calcium determination, in particular to a freeze-dried reagent ball, a biochemical analysis chip and a serum total calcium determination method. The embodiment of the invention provides a freeze-dried reagent ball, a biochemical analysis chip and a serum total calcium determination method, wherein the freeze-dried reagent ball is prepared by freeze-drying liquid drops of a serum total calcium determination reagent; the serum total calcium determination reagent comprises buffer solution 40-200mmol/L, potassium chloride 40-200mmol/L, 8-hydroxyquinoline-5-sulfonic acid 5-100mmol/L, azoarsenic III 0.5-2mmol/L and excipient 0.3-60 wt%; wherein the pH value of the buffer solution is 7.5-9.5. Therefore, the concentration of each reactant in the serum total calcium determination reagent is changed to change the concentration of the reactant in the freeze-dried reagent ball and change the determination wavelength, so that the reagent absorbance value in the test is reduced, the test precision and accuracy of the freeze-dried reagent ball, the biochemical analysis chip and the serum total calcium determination method are improved, and the determination range is widened.
Description
Technical Field
The invention relates to the technical field of serum total calcium determination, in particular to a freeze-dried reagent ball, a biochemical analysis chip and a serum total calcium determination method.
Background
Calcium ion is one of the indispensable ions for maintaining the normal operation of various physiological activities of the body. For example, calcium ions are capable of maintaining biopotentials across cell membranes, as well as maintaining normal nerve conduction function. The increase of serum total calcium is mostly seen in hyperparathyroidism, multiple myeloma and sarcoidosis, and excessive calcium absorption in the intestinal tract caused by the large-scale application of vitamin treatment. Decreased serum total calcium may cause increased neuromuscular excitability and tetany, which is often seen in infantile tetany, vitamin deficiency, diseases causing decreased serum protein, and chronic renal failure.
In the prior art, common determination methods for serum total calcium include colorimetry, flame photometry, atomic absorption spectrophotometry, titration, radionuclide dilution mass spectrometry and the like. However, the existing reagent for measuring total calcium in serum has the problems of low test precision, large accuracy deviation and over-narrow detection range in a biochemical analysis chip.
Disclosure of Invention
In order to solve the problems of low test precision, large accuracy deviation and over-narrow detection range of the conventional serum total calcium determination reagent in a biochemical analysis chip, the embodiment of the invention provides a freeze-dried reagent ball, a determination chip and a determination method, which can reduce the reagent test absorbance value and change the determination wavelength by changing the concentration of reactants in the freeze-dried reagent ball so as to improve the test precision and accuracy of the serum total calcium determination reagent and widen the determination range.
In order to solve the above technical problem, an embodiment of the present invention provides the following technical solutions:
in a first aspect, the embodiments of the present invention provide a freeze-dried reagent ball for measuring serum total calcium, the freeze-dried reagent ball is made by freeze-drying a droplet of a serum total calcium measuring reagent,
the serum total calcium determination reagent comprises the following components:
40-200mmol/L of buffer solution, 40-200mmol/L of potassium chloride, 5-100mmol/L of 8-hydroxyquinoline-5-sulfonic acid, 0.5-2mmol/L of azoarsenic III and 0.3-60 wt% of excipient; wherein the content of the first and second substances,
the pH value of the buffer solution is 7.5-9.5.
Optionally, the excipient comprises monosaccharide and/or disaccharide, and the excipient further comprises sodium cholate and dextran;
the total weight of the monosaccharide and the disaccharide accounts for 0.1 to 20 weight percent of the total weight of the serum calcium determination reagent;
the weight percentage of the sodium cholate in the total calcium serum determination reagent is 0.1-20 wt%;
the weight percentage of the glucan in the total serum calcium determination reagent is 0.1-20 wt%.
Optionally, the buffer comprises: one or more of a tris buffer, a boric acid buffer, a glycine buffer, a citric acid/trisodium citrate buffer, and a phosphoric acid/sodium phosphate buffer.
Optionally, the buffer solution is tris buffer solution, the disaccharide is trehalose, and the glucan is 1 ten thousand glucan;
the serum total calcium determination reagent comprises the following components:
40mmol/L of tris buffer solution, 200mmol/L of potassium chloride, 10mmol/L of 8-hydroxyquinoline-5-sulfonic acid, 1.25mmol/L of azoarsenic III, 0.5 wt% of trehalose, 3 wt% of sodium cholate and 1 ten thousand 1 wt% of glucan.
Optionally, the buffer solution is glycine buffer solution, the monosaccharide is glucose, and the glucan is 4 ten thousand glucan;
the serum total calcium determination reagent comprises the following components:
40mmol/L of glycine buffer solution, 50mmol/L of potassium chloride, 8-hydroxyquinoline-5-sulfonic acid: 70mmol/L, 2.00mmol/L of azoarsenic III, 15 wt% of glucose, 0.2 wt% of sodium cholate and 4 ten thousand of glucan 1.8 wt%.
Optionally, the buffer is a boric acid buffer, the disaccharide is sucrose, and the glucan is 2 ten thousand glucan;
the serum total calcium determination reagent comprises the following components:
100mmol/L boric acid buffer solution, 30mmol/L potassium chloride, 18 mmol/L11-hydroxyquinoline-5-sulfonic acid, 0.5mmol/L azoarsenic III, 12 wt% sucrose, 3 wt% sodium cholate and 2 ten thousand 1 wt% dextran.
Optionally, the volume of the drop of serum total calcium determination reagent is 3.0 μ l-4.0 μ l.
In a second aspect, an embodiment of the present invention provides a biochemical analysis chip, including a chip body and the lyophilized reagent ball according to the first aspect, wherein the chip body is provided with a colorimetric hole, and the lyophilized reagent ball is accommodated in the colorimetric hole.
In a third aspect, the present invention also provides a method for measuring total serum calcium, which is applied to the biochemical analysis chip according to the second aspect, and the method includes:
diluting the serum sample by a diluent;
filling the diluted serum sample into the colorimetric hole;
after the diluted serum sample and the freeze-dried reagent ball in the colorimetric hole fully react, testing the absorbance value of the solution in the colorimetric hole by using a spectrophotometer; wherein the content of the first and second substances,
the wavelength of the light source generated by the spectrophotometer comprises a main wavelength and a secondary wavelength, wherein the main wavelength is 500-700nm, and the secondary wavelength is 750-850 nm.
Optionally, the primary wavelength is one of 546nm, 605nm, 630nm, 670nm, or 700nm, and the secondary wavelength is 800 nm.
The beneficial effects of the embodiment of the invention are as follows: different from the situation of the prior art, the embodiment of the invention provides a freeze-dried reagent ball, a biochemical analysis chip and a serum total calcium determination method, wherein the freeze-dried reagent ball is prepared by freeze-drying liquid drops of a serum total calcium determination reagent; the serum total calcium determination reagent comprises buffer solution 40-200mmol/L, potassium chloride 40-200mmol/L, 8-hydroxyquinoline-5-sulfonic acid 5-100mmol/L, azoarsenic III 0.5-2mmol/L and excipient 0.3-60 wt%; wherein the pH value of the buffer solution is 7.5-9.5. Therefore, the concentration of each reactant in the serum total calcium determination reagent is changed to change the concentration of the reactant in the freeze-dried reagent ball and change the determination wavelength, so that the reagent absorbance value in the test is reduced, the test precision and accuracy of the freeze-dried reagent ball, the biochemical analysis chip and the serum total calcium determination method are improved, and the determination range is widened.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required to be used in the embodiments of the present invention will be briefly described below. It is obvious that the drawings described below are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.
FIG. 1 is a schematic flow chart of a method for measuring total serum calcium according to an embodiment of the present invention;
FIG. 2 is a diagram showing the results of clinical correlation analysis using the biochemical analysis chip according to an embodiment of the present invention;
FIG. 3 is a graph of the results of a linear range test provided by one embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It should be noted that, if not conflicted, the various features of the embodiments of the invention may be combined with each other within the scope of protection of the invention. Additionally, while functional block divisions are performed in the device diagrams, with logical sequences shown in the flowcharts, in some cases, the steps shown or described may be performed in a different order than the block divisions in the device diagrams, or the flowcharts.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The embodiment of the invention provides a freeze-dried reagent ball for measuring total calcium in human serum, which is prepared by freeze-drying liquid drops of a total calcium measurement reagent in serum. Specifically, the serum total calcium determination reagent comprises the following components: 40-200mmol/L of buffer solution, 40-200mmol/L of potassium chloride, 5-100mmol/L of 8-hydroxyquinoline-5-sulfonic acid, 0.5-2mmol/L of azoarsenic III and 0.3-60 wt% of excipient.
In some embodiments, the serum total calcium assay reagents are formulated as follows: measuring a certain volume of buffer solution, and adding the buffer solution into a container filled with 800ml of water; after the buffer solution is completely dissolved in water, adding a certain amount of potassium chloride, 8-hydroxyquinoline-5-sulfonic acid and azoarsenic III into a container; adding excipient into the container; finally, water was added to the vessel to bring the volume of the solution in the vessel to 1L.
In some embodiments, the method of preparing a lyophilized reagent pellet comprises the steps of:
s11, dropping the liquid drops of the serum total calcium determination reagent in liquid nitrogen to form ice balls;
for example, the liquid drop of the serum total calcium determination reagent can be dropped in liquid nitrogen by a dispenser, and the liquid drop of the serum total calcium determination reagent can be coagulated into an ice ball in the liquid nitrogen. The size of the liquid drop of the mixed liquid dropped into the liquid nitrogen can be adjusted by those skilled in the art according to actual needs, and the volume of the ice ball can be adjusted by controlling the size of the liquid drop. Alternatively, in some embodiments, the volume of serum total calcium assay reagents is 3.0. mu.l to 4.0. mu.l.
S14, freeze-drying the ice ball to obtain a freeze-dried reagent ball;
in this embodiment, the ice ball is placed in a vacuum freeze dryer to be freeze-dried to form a freeze-dried reagent ball, and the freeze-dried reagent ball is collected and stored in a dry aluminum bottle after being repressed by nitrogen gas.
The freeze drying is a drying method which is to freeze the serum total calcium determination reagent containing water into solid by cooling in advance, and dehydrate the serum total calcium determination reagent at low temperature by utilizing the sublimation performance of water under the condition of low temperature and reduced pressure so as to achieve the drying purpose. After freeze-drying, the buffer solution, potassium chloride, 8-hydroxyquinoline-5-sulfonic acid, azoarsenic III and the excipient in the serum total calcium determination reagent are left in an ice shelf during freezing, so that the freeze-dried reagent ball after freeze-drying is loose and porous, and the volume of the freeze-dried reagent ball is basically unchanged from that before freeze-drying. Because the freeze-dried reagent ball is always in a frozen state before being dried, and the ice crystals are uniformly distributed in each component of the serum total calcium determination reagent. In the sublimation process, the components are not concentrated due to dehydration. Therefore, the freeze-dried reagent ball after freeze drying is spongy, loose and porous, and is easy to dissolve in water and restore to the original shape.
The buffer solution is a mixed solution composed of weak acid and salt thereof, weak base and salt thereof, and can offset or reduce the influence of external strong acid or strong base on the pH value of the solution to a certain extent, so that the pH value of the solution is kept relatively stable. The buffer solution in the embodiment of the present invention includes one or more of Tris buffer solution, boric acid buffer solution, glycine buffer solution, citric acid/trisodium citrate buffer solution, and phosphoric acid/sodium phosphate buffer solution. Wherein, the citric acid/trisodium citrate buffer solution is a mixed solution of citric acid and trisodium citrate, and the phosphoric acid/sodium phosphate buffer solution is a buffer solution of phosphoric acid and sodium phosphate.
In the embodiment of the invention, the buffer solution is an alkaline buffer solution, and the pH value of the buffer solution is 7.5-9.5. Since the isoelectric point of most proteins in serum is in the acidic range, proteins readily form precipitates in acidic buffers to interfere with calcium determination. On the other hand, under alkaline conditions, although the interference caused by protein precipitation can be avoided, the magnesium ions in serum are likely to react with azoarsenIII to form a complex, which causes interference. Therefore, in order to avoid protein precipitation and interference caused by magnesium ions, 8-hydroxyquinoline-5-sulfonic acid, a magnesium ion masking agent, is added to the alkaline buffer.
The excipient can endow the freeze-dried reagent ball with good appearance, so that the freeze-dried reagent ball is loose and porous and is easy to redissolve. The excipient mainly comprises polyalcohol, saccharide, amino acids, inorganic salts, protein and peptide excipient. The polyalcohol excipient comprises glycerol, sorbitol, mannitol, inositol, adonitol, ethylene glycol, polyethylene glycol, etc. The saccharide excipient comprises monosaccharide excipient, disaccharide excipient and polysaccharide excipient; wherein the monosaccharide excipient comprises glucose; disaccharide excipients include sucrose, lactose, maltose, trehalose, and the like; the polysaccharide excipient comprises water soluble starch, maltodextrin, and dextran, wherein the dextran can be one or more of dextran 1 ten thousand, dextran 2 ten thousand, dextran 4 ten thousand or dextran 7 ten thousand. Amino acid excipients include: sodium glutamate, proline, lysine, alanine, and the like; inorganic salt excipients include: phosphates, calcium carbonate, manganese sulfate, sodium cholate, sodium acetate and the like; protein and peptide excipients include mucopolysaccharide protein, casein, bovine serum albumin, and the like.
In order to improve the appearance of the freeze-dried reagent ball and improve the dissolving effect of the freeze-dried reagent ball, the excipient in the embodiment of the invention also comprises monosaccharide and/or disaccharide besides sodium cholate and dextran; and the total weight of the monosaccharide and the disaccharide accounts for 0.1 to 20 percent of the weight of the serum total calcium determination reagent; the weight of the sodium cholate and the weight of the glucan both account for 0.1 to 20 percent of the weight of the serum total calcium determination reagent. The freeze-dried reagent ball provided by the embodiment of the invention is suitable for large-scale batch production, and the solid freeze-dried reagent ball is more convenient to store and transport.
The embodiment of the invention provides a freeze-dried reagent ball test for determining the content of total calcium in serum, which has the following determination principle: under the alkaline condition, 8-hydroxyquinoline-5 sulfonic acid is utilized to mask magnesium ions, azoarsenic III reacts with calcium ions to form a purple complex, so that the reaction liquid has an absorption peak at a certain wavelength, and the absorbance value of the reaction liquid at the wavelength is in direct proportion to the concentration of the calcium ions within a certain range. Therefore, the concentration of calcium ion in the serum sample can be calculated according to the rising rate of the absorbance.
The embodiment of the invention also provides a biochemical analysis chip which comprises a chip body and the freeze-drying reagent ball. The chip body is provided with a colorimetric hole, and the freeze-dried reagent ball is accommodated in the colorimetric hole.
Referring to fig. 1, fig. 1 is a schematic flow chart of a method for measuring total serum calcium according to an embodiment of the present invention, and as shown in fig. 1, the method includes the following steps:
s11, diluting the serum sample by a diluent;
s12, filling the diluted serum sample into the colorimetric hole;
and S13, after the serum sample to be diluted fully reacts with the freeze-dried reagent ball in the colorimetric hole, testing the absorbance value of the solution in the colorimetric hole by using a spectrophotometer.
In some embodiments, the biochemical analysis chip further comprises a sample well, a diluent well, a sample quantification well, a diluent quantification well, and a mixing well. When the biochemical analysis chip is used for testing, the sample can be detected without centrifugal treatment, so that serum, plasma and whole blood can be used for detection. The serum sample is added to the sample well from the sample well by a pipette, and the diluent is added to the diluent well from the diluent well by a pipette. And enabling the diluent in the diluent quantifying groove and the serum sample in the sample quantifying groove to enter a mixing groove so as to dilute the serum sample through the diluent. And filling the diluted serum sample in the mixing tank into the colorimetric hole in a centrifugal mode, and testing the absorbance of the solution in the colorimetric hole by using a spectrophotometer after the diluted serum sample and the freeze-dried reagent ball in the colorimetric hole fully react.
Specifically, in the above example, the diluent may be distilled water. The wavelength of the light source generated by the spectrophotometer comprises a main wavelength and a secondary wavelength, wherein the main wavelength of the light source is 500-700nm, and the secondary wavelength of the light source is 750-850 nm. In order to improve the test precision and widen the test range, the main wavelength of the light source can be one of 546nm, 605nm, 630nm, 670nm or 700nm, and the auxiliary wavelength is 800 nm.
The embodiment of the invention provides a freeze-dried reagent ball, a biochemical analysis chip and a serum total calcium determination method, wherein the freeze-dried reagent ball is prepared by freeze-drying liquid drops of a serum total calcium determination reagent; the serum total calcium determination reagent comprises buffer solution 40-200mmol/L, potassium chloride 40-200mmol/L, 8-hydroxyquinoline-5-sulfonic acid 5-100mmol/L, azoarsenic III 0.5-2mmol/L and excipient 0.3-60 wt%; wherein the pH value of the buffer solution is 7.5-9.5. Therefore, the concentration of each reactant in the serum total calcium determination reagent is changed to change the concentration of the reactant in the freeze-dried reagent ball and change the determination wavelength, so that the reagent absorbance value in the test is reduced, the test precision and accuracy of the freeze-dried reagent ball, the biochemical analysis chip and the serum total calcium determination method are improved, and the determination range is widened.
To further illustrate the technical solutions of the present invention, several examples of the preparation method of the serum total calcium determination reagent of the present invention are provided below.
Example 1:
the serum total calcium determination reagent comprises the following components:
40mmol/L of tris buffer solution, 200mmol/L of potassium chloride, 10mmol/L of 8-hydroxyquinoline-5-sulfonic acid, 1.25mmol/L of azoarsenic III, 0.5 wt% of trehalose, 3 wt% of sodium cholate and 1 ten thousand 1 wt% of glucan; wherein the pH value of the tris buffer is 8.0.
In this example, the lyophilized reagent beads were prepared by the above method, and the volume of the reagent droplet for measuring total serum calcium was about 3.5 ul.
The biochemical analysis chip in this embodiment includes a chip body and the freeze-dried reagent ball provided in this embodiment. The method for measuring the total calcium in the serum is adopted to detect the absorbance value of a serum sample, and the main wavelength of a light source generated by a spectrophotometer is 630nm and the auxiliary wavelength is 800nm during the test.
Example 2:
the present example differs from example 1 as follows:
the serum total calcium determination reagent comprises the following components:
40mmol/L of glycine buffer solution, 50mmol/L of potassium chloride, 8-hydroxyquinoline-5-sulfonic acid: 70mmol/L of azo arsenic III 2.00mmol/L, 15 wt% of glucose, 0.2 wt% of sodium cholate and 4 ten thousand of glucan 1.8 wt%; wherein the pH value of the glycine buffer solution is 9.0.
The spectrophotometer used for the test produced a dominant wavelength of the light source of 670 nm.
Example 3
This example differs from examples 1 and 2 in that:
the serum total calcium determination reagent comprises the following components:
100mmol/L boric acid buffer solution, 30mmol/L potassium chloride, 18 mmol/L11-hydroxyquinoline-5-sulfonic acid, 0.5mmol/L azoarsenic III, 12 wt% sucrose, 3 wt% sodium cholate and 2 ten thousand 1 wt% dextran; wherein the pH value of the boric acid buffer solution is 7.5.
The spectrophotometer used for the test produced a dominant wavelength of the light source of 546 nm.
The performance of the biochemical analysis chip obtained in example 1 of the present invention will be described below with reference to tables. The change value of absorbance of the measurement chip injected with the serum sample at 37 ℃ was measured by a vp10 portable biochemical analyzer manufactured by Shenzhen Jinrui Bio Inc. The serum total calcium concentration in the serum samples was calculated using calibration standards provided by the british langway company.
1) And testing precision: the biochemical analysis chip provided in the embodiment 1 of the invention is used for testing the serum total calcium concentration of a serum sample with the known serum total calcium concentration of 2.18mmol/L for 20 times, and the average value of the concentration values is calculated by the following formula
Standard Deviation (SD) and Coefficient of Variation (CV):
wherein, XiThe concentration value was measured for the i-th time, and n is the number of times of measurement.
Obtaining the average value of the measured concentration values obtained by testing the same serum sample for 20 times
2.24mmol/L, standard deviation SD 0.0463, coefficient of variation CV 2.07%. Generally, a larger standard deviation represents a larger difference between most of the test results and their average values; if the standard deviation is small, it means that the test results are closer to the average.
2) And testing the accuracy: the biochemical analysis chip provided by the embodiment 1 of the invention is adopted to test a serum sample with the known serum total calcium concentration of 2.13mmol/L for three times, the measured serum total calcium concentration value is obtained, the average value of the serum total calcium concentration values measured for 3 times is calculated to be 2.16mmol/L, and the relative deviation is 1.74%.
3) And specificity test:
in the serum sample A, the actual concentration of calcium ion is known to be 2.26mmol/L, and the serum sample A contains 50mg/dl ascorbic acid. In the serum sample B, it is known that the true concentration of calcium ion is 2.03mmol/L, and the serum sample B contains 30mg/dl bilirubin. In the serum sample C, it is known that the true concentration of calcium ion is 2.35mmol/L, and the serum sample C contains hemoglobin 500 mg/dl. The test results are shown in the following table:
as can be seen from the above table, although ascorbic acid, bilirubin or hemoglobin is added to the serum sample, the presence of ascorbic acid, bilirubin or hemoglobin has a very slight effect on the test results of the biochemical analysis chip.
4) And clinical relevance analysis:
the total calcium content in serum of a plurality of serum samples with different calcium ion concentrations is simultaneously measured by adopting the biochemical analysis chip and the Abaxis reagent disk provided by the embodiment 1 of the invention. The corresponding measured serum total calcium concentration value (unit mmol/L) is shown in FIG. 2, the X column is the serum total calcium concentration measured by the Abaxis reagent disk, and the Y column is the serum total calcium concentration measured by the biochemical analysis chip provided in example 1 of the present invention.
The correlation equation between the two groups of test results of the biochemical analysis chip and the Abaxis reagent disk provided by the embodiment of the invention is as follows:
y＝0.9002x+0.2683
coefficient of correlation R2The closer the correlation coefficient is to 1, the stronger the correlation between the two sets of data, 0.9953. Therefore, the correlation between the test results of the biochemical analysis chip provided by the embodiment of the invention and the test results of the Abaxis reagent disk is strong.
5) Linear range test
The test method is as follows: serum samples at 6 dilution concentrations were mixed as shown in table two using high concentration (active) samples near the upper end of the linear range and low concentration (active) samples near the lower end of the linear range.
Table two:
| |
1 | 2 | 3 | 4 | 5 | 6 |
| High concentration (active) |
0 portion of | 1 part of | 2 portions of | 3 portions of | 4 portions of | 5 portions of |
| Low concentration (active) samples | 5 portions of | 4 portions of | 3 portions of | 2 portions of | 1 part of | 0 portion of |
The biochemical analysis chip provided by the embodiment 1 of the invention is adopted to respectively test the serum total calcium concentration of 6 serum samples, each serum sample is tested for 3 times, and the average value (y) of the concentration values measured by the serum total calcium in the 6 serum samples is respectively calculatedi). Concentration (x) after dilution with each samplei) As independent variable, mean value of measured concentration values (y) of each samplei) Linear regression equations were solved for the dependent variables. Calculating a correlation coefficient R of the linear regression according to a formula (4); equation (4) is as follows:
wherein n is of the measurement sampleNumber, xiTo dilute the concentration, yiThe average value of the measurement results is shown.
As shown in fig. 3, the linear regression equation obtained is y 0.8861x +0.2417, and the correlation coefficient R is2＝0.9951。
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; within the idea of the invention, also technical features in the above embodiments or in different embodiments may be combined, steps may be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A freeze-dried reagent ball for measuring serum total calcium is characterized in that the freeze-dried reagent ball is prepared by freeze-drying liquid drops of a serum total calcium measuring reagent,
the serum total calcium determination reagent comprises the following components:
40-200mmol/L of buffer solution, 40-200mmol/L of potassium chloride, 5-100mmol/L of 8-hydroxyquinoline-5-sulfonic acid, 0.5-2mmol/L of azoarsenic III and 0.3-60 wt% of excipient; wherein the content of the first and second substances,
the pH value of the buffer solution is 7.5-9.5.
2. The lyophilized reagent ball of claim 1, wherein the excipient comprises a monosaccharide and/or disaccharide, and the excipient further comprises sodium cholate and dextran;
the total weight of the monosaccharide and the disaccharide accounts for 0.1 to 20 weight percent of the total weight of the serum calcium determination reagent;
the weight percentage of the sodium cholate in the total calcium serum determination reagent is 0.1-20 wt%;
the weight percentage of the glucan in the total serum calcium determination reagent is 0.1-20 wt%.
3. The lyophilized reagent ball of claim 1, wherein the buffer comprises: one or more of a tris buffer, a boric acid buffer, a glycine buffer, a citric acid/trisodium citrate buffer, and a phosphoric acid/sodium phosphate buffer.
4. The lyophilized reagent ball of claim 2, wherein the buffer is tris buffer, the disaccharide is trehalose, and the dextran is 1 ten thousand dextran;
the serum total calcium determination reagent comprises the following components:
40mmol/L of tris buffer solution, 200mmol/L of potassium chloride, 10mmol/L of 8-hydroxyquinoline-5-sulfonic acid, 1.25mmol/L of azoarsenic III, 0.5 wt% of trehalose, 3 wt% of sodium cholate and 1 ten thousand 1 wt% of glucan.
5. The lyophilized reagent ball of claim 2, wherein the buffer is glycine buffer, the monosaccharide is glucose, and the dextran is dextran 4 ten thousand;
the serum total calcium determination reagent comprises the following components:
40mmol/L of glycine buffer solution, 50mmol/L of potassium chloride, 8-hydroxyquinoline-5-sulfonic acid: 70mmol/L, 2.00mmol/L of azoarsenic III, 15 wt% of glucose, 0.2 wt% of sodium cholate and 4 ten thousand of glucan 1.8 wt%.
6. The lyophilized reagent ball of claim 2, wherein the buffer is a boric acid buffer, the disaccharide is sucrose, and the dextran is 2 ten thousand dextran;
the serum total calcium determination reagent comprises the following components:
100mmol/L boric acid buffer solution, 30mmol/L potassium chloride, 18 mmol/L11-hydroxyquinoline-5-sulfonic acid, 0.5mmol/L azoarsenic III, 12 wt% sucrose, 3 wt% sodium cholate and 2 ten thousand 1 wt% dextran.
7. The lyophilized reagent pellet according to any one of claims 1 to 6, wherein the volume of the droplet of the serum total calcium assay reagent is 3.0 μ l to 4.0 μ l.
8. A biochemical analysis chip, characterized in that, the biochemical analysis chip comprises a chip body and a freeze-dried reagent ball according to any one of claims 1 to 6, the chip body is provided with a colorimetric hole, and the freeze-dried reagent ball is accommodated in the colorimetric hole.
9. A method for measuring total serum calcium, which is applied to the biochemical analysis chip according to claim 8, wherein the method comprises:
diluting the serum sample by a diluent;
filling the diluted serum sample into the colorimetric hole;
after the diluted serum sample and the freeze-dried reagent ball in the colorimetric hole fully react, testing the absorbance value of the solution in the colorimetric hole by using a spectrophotometer; wherein the content of the first and second substances,
the wavelength of the light source generated by the spectrophotometer comprises a main wavelength and a secondary wavelength, wherein the main wavelength is 500-700nm, and the secondary wavelength is 750-850 nm.
10. The method for measuring total serum calcium according to claim 9, wherein the primary wavelength is one of 546nm, 605nm, 630nm, 670nm and 700nm, and the secondary wavelength is 800 nm.
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