CN112683892A - Calibration color card for biochemical detector and preparation method thereof - Google Patents
Calibration color card for biochemical detector and preparation method thereof Download PDFInfo
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- IICCLYANAQEHCI-UHFFFAOYSA-N 4,5,6,7-tetrachloro-3',6'-dihydroxy-2',4',5',7'-tetraiodospiro[2-benzofuran-3,9'-xanthene]-1-one Chemical compound O1C(=O)C(C(=C(Cl)C(Cl)=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 IICCLYANAQEHCI-UHFFFAOYSA-N 0.000 claims abstract description 8
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of chemical analysis, and particularly relates to a calibration color card for a biochemical detector and a preparation method thereof. The invention provides a calibration color card, comprising: the color card comprises a color card shell and a calibration color card paper arranged in a calibration area of the color card shell; the calibration color card paper comprises chromatography paper and a calibration color layer arranged on the surface of the chromatography paper, wherein the calibration color layer contains a red coloring agent and a blue coloring agent; the red coloring agent comprises one or more of fuchsin, rose bengal, rhodamine B and fluorescein; the blue coloring agent comprises one or more of indigo disulfonic acid sodium, methine blue, Prussian blue and bromophenol blue. Experimental results show that the reflecting surface of the calibration color chart provided by the invention is very close to the surface of a saccharified blood sample formed on chromatographic paper, and the reactivity of a saccharified reflecting system of a biochemical detector can be effectively calibrated.
Description
Technical Field
The invention belongs to the field of chemical analysis, and particularly relates to a calibration color card for a biochemical detector and a preparation method thereof.
Background
In POCT biochemical detectors, the detection of the characteristic or concentration of glycated hemoglobin in a blood sample is often performed by detecting the intensity of red light and blue light scattered by a reactant. For example, a glycated hemoglobin biochemical analyzer for measuring the content of glycated hemoglobin in blood by using a boric acid affinity chromatography. The principle of detection of glycated hemoglobin by boronic acid affinity chromatography is that glycated hemoglobin (HBA1c) specifically complexes with boronic acid to exhibit a blue color, whereas non-glycated hemoglobin (Hb) does not react with boronic acid and still exhibits its original color, red. The relative content of the glycosylated hemoglobin can be calculated by respectively measuring the light intensity of the red light and the blue light reflected by the sample through the color reflection module.
Before the biochemical detector enters clinical test, the most important step is to calibrate the inter-platform difference so as to ensure that the detection precision of all the instruments is at the same level and within the range required by industry standard. The inter-apparatus difference is defined as the difference between different apparatuses testing the same sample, and is usually evaluated by the Coefficient of Variation (CV) of the test value of the same sample in different apparatuses. Due to the careful use of biological samples for instrument calibration in mass production, and the high skill requirements of the biological samples for the calibration operator. Therefore, a non-biological calibration method is imperative, and the time, labor and material cost for calibrating the instrument can be greatly saved.
For the detection of the glycosylated hemoglobin, the current standard in China is YY/T1246-. The "calibration standards for glycated hemoglobin analyzers" are currently being established by the national Committee for Biometrics and are seeking industry standards for enrichment. Each unit of measure can be added to the standard specification according to the relevant standard forming calibration method.
The conventional glycated hemoglobin analyzer mainly uses a glycated hemoglobin standard substance as a standard. At present, the national standard substance resource sharing platform uses standard substances taking frozen human hemoglobin as a main component to carry out calibration reference of a glycosylated hemoglobin analyzer. This is a biological sample, with the risk of use. In addition, in practical use, standard substances which do not meet the requirements can be used, so that the linear correlation cannot be calibrated according to the existing standard in practical operation.
Disclosure of Invention
The present invention is directed to a calibration color chart for a biochemical detector and a method for manufacturing the same, wherein the calibration color chart does not contain biological components and is suitable for calibrating a biochemical detector using red and blue light for detection, such as a boric acid affinity chromatography glycated hemoglobin detector.
The invention provides a calibration color card for a biochemical detector, which comprises: the color card comprises a color card shell and a calibration color card paper arranged in a calibration area of the color card shell;
the calibration color card paper comprises chromatography paper and a calibration color layer arranged on the surface of the chromatography paper, wherein the calibration color layer contains a red coloring agent and a blue coloring agent;
the red coloring agent comprises one or more of fuchsin, rose bengal, rhodamine B and fluorescein;
the blue coloring agent comprises one or more of indigo disulfonic acid sodium, methine blue, Prussian blue and bromophenol blue.
Preferably, the red staining agent is fluorescein; the blue colorant is methine blue.
Preferably, the chromatographic paper comprises a retention layer, a water stop layer and an adsorption layer which are contacted in sequence, wherein the retention layer is positioned on the top of the chromatographic paper.
The invention provides a preparation method of a calibration color card for a biochemical detector, which comprises the following steps:
a) preparing a mixed dyeing solution containing a red dyeing agent and a blue dyeing agent, dripping the mixed dyeing solution onto the surface of the chromatographic paper, and forming a calibration color layer on the surface of the chromatographic paper;
the red coloring agent comprises one or more of fuchsin, rose bengal, rhodamine B and fluorescein; the blue coloring agent comprises one or more of indigo disulfonic acid sodium, methine blue, Prussian blue and bromophenol blue;
b) and (3) filling the chromatographic paper with the calibration color layer into the calibration area groove of the color card shell, and buckling the calibration area cover plate of the color card shell to obtain the calibration color card for the biochemical detector.
Preferably, in the step a), the mixed dyeing solution is prepared by mixing a red dyeing solution and a blue dyeing solution;
the concentration of the coloring agent in the red coloring agent solution is 2-10 g/50 mL; the concentration of the coloring agent in the blue coloring agent solution is 150-250 mg/50 mL.
Preferably, the red stain solution is an alcohol solution of fluorescein; the blue colorant solution is an aqueous solution of methylene blue.
Preferably, the alcohol solution of fluorescein is prepared by the following steps:
and mixing the fluorescein crude product with alcohol, and filtering to remove impurities to obtain an alcohol solution of the fluorescein.
Preferably, the mixing ratio of the red dye solution and the blue dye solution is determined according to the reactivity of the calibration color card to be prepared; the higher the reactivity of the calibration color chart to be prepared, the higher the proportion of the blue colorant solution.
Preferably, in the step a), when the mixed staining solution is dripped, a restraining tool for restraining the diffusion of the staining solution is arranged on the chromatographic paper;
the restraint frock has big end down's circular through-hole, and the draft of through-hole is 5 ~ 15.
Preferably, the method further comprises the following steps: baking the calibration color card obtained in the step b).
Compared with the prior art, the invention provides a calibration color card for a biochemical detector and a preparation method thereof. The invention provides a calibration color card, comprising: the color card comprises a color card shell and a calibration color card paper arranged in a calibration area of the color card shell; the calibration color card paper comprises chromatography paper and a calibration color layer arranged on the surface of the chromatography paper, wherein the calibration color layer contains a red coloring agent and a blue coloring agent; the red coloring agent comprises one or more of fuchsin, rose bengal, rhodamine B and fluorescein; the blue coloring agent comprises one or more of indigo disulfonic acid sodium, methine blue, Prussian blue and bromophenol blue. According to the invention, the coloring agents with red and blue colors and solid granularity close to hemoglobin are selected and uniformly deposited on the surface of the chromatographic paper, so that the color card paper without biological components is obtained; the surface appearance of the color card paper is similar to the surface appearance formed after the surface affinity chromatography of the saccharified blood sample on the surface of the chromatographic paper, and the color card paper can be made into a standard sample for calibrating the total reactivity of the saccharified reflecting system of the biochemical detector. The invention installs the color card paper developed and designed based on the technical conception into the color card shell, thereby assembling and obtaining the calibration color card without biological components, and the calibration color card can be used for calibrating a biochemical detector which adopts red and blue light to detect, such as a boric acid affinity chromatography glycosylated hemoglobin detector. Experimental results show that the reflecting surface of the calibration color chart provided by the invention is very close to the surface of a saccharified blood sample formed on chromatographic paper, and the reactivity of a saccharified reflecting system of a biochemical detector can be effectively calibrated.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a calibration color chip for a biochemical detector according to an embodiment of the present invention;
FIG. 2 is a schematic view of a color chip housing provided with a calibration area cover plate not assembled according to an embodiment of the present invention;
FIG. 3 is a schematic structural diagram of a cover plate of the calibration area according to an embodiment of the present invention;
FIG. 4 is a schematic structural diagram of a three-layer chromatography paper provided by an embodiment of the invention;
FIG. 5 is a schematic view of a retention layer structure provided in an embodiment of the invention;
FIG. 6 is a schematic diagram of a process for trapping a stain and forming a colored surface in a retention layer of a chromatography paper provided by an embodiment of the invention;
FIG. 7 is a schematic diagram of an operation of dropping a mixed staining solution on a chromatographic paper provided with a constraint fixture according to an embodiment of the present invention;
FIG. 8 is a schematic structural diagram of a constraint fixture provided in an embodiment of the present invention;
FIG. 9 is a comparison of a calibration color chip reflective surface and a blood sample reflective surface at 20 times microscope provided by an embodiment of the present invention;
FIG. 10 is a calibration graph of tester reactivity versus standard reactivity according to an embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a calibration color card for a biochemical detector, which comprises: the color card comprises a color card shell and a calibration color card paper arranged in a calibration area of the color card shell;
the calibration color card paper comprises chromatographic paper and a calibration color layer arranged on the surface of the chromatographic paper, wherein the calibration color layer contains a red coloring agent and a blue coloring agent.
Referring to fig. 1, fig. 1 is a schematic structural diagram of a calibration color chart for a biochemical detector according to an embodiment of the present invention, where 1 is a color chart housing, 1-1 is a calibration area cover plate, and 2 is a calibration color chart paper.
The calibration colour chip provided by the invention comprises a colour chip shell 1 and a calibration colour chip card 2. The color chip shell 1 is used for installing and fixing the calibration color chip card 2, and the specific structure of the color chip shell comprises a color chip shell main part and a calibration area cover plate 1-1 which is detachable from the color chip shell main part, the color chip shell main part is provided with a calibration area for placing the calibration color chip card 2, and the calibration area cover plate 1-1 is used for fixing and protecting the calibration color chip card 2 placed in the calibration area. In the present invention, the calibration area cover plate 1-1 is a hollow structure, and the hollow area is used to expose a detection area (i.e., a reflection surface) of the calibration color jam paper 2. In the present invention, the specific shape and structure of the color chip housing 1 depend on the applicable detector. In the embodiment of the present invention, a specific structure of the adopted color chart housing 1 is as shown in fig. 2, fig. 2 is a schematic structural view of the color chart housing without assembling the calibration area cover plate according to the embodiment of the present invention, wherein the left drawing is a front view, the right drawing is a side view, and 1-2 are calibration color chart paper mounting positions (i.e., calibration areas). In the embodiment provided by the present invention, a specific structure of the adopted calibration area cover plate 1-1 is shown in fig. 3, and fig. 3 is a schematic structural view of the calibration area cover plate provided by the embodiment of the present invention, wherein the upper left drawing is a front view, the lower left drawing is a side view, and the right drawing is a cross-sectional view.
In the calibration color chart provided by the present invention, the calibration color chart paper 2 is installed in the calibration area of the color chart housing 1, and the calibration color chart paper includes a chromatography paper and a calibration color layer provided on the surface of the chromatography paper. Wherein the calibration color layer contains a red colorant and a blue colorant; the red coloring agent comprises one or more of fuchsin, rose bengal, rhodamine B and fluorescein, and is preferably fluorescein; the blue coloring agent comprises one or more of indigo disulfonic acid sodium, methine blue, Prussian blue and bromophenol blue, and is preferably methine blue. In the invention, the reactivity value of the provided calibration color card is directly related to the content of the red coloring agent and the blue coloring agent in the calibration color card paper, and a series of calibration color cards with different reactivities can be obtained by adjusting the content of the two coloring agents.
In the calibration color card provided by the invention, the chromatography paper is preferably of a three-layer structure, the specific structure of which is shown in fig. 4-5, fig. 4 is a schematic structural diagram of the chromatography paper of the three-layer structure provided by the embodiment of the invention, wherein 2-1 is a retention layer, 2-2 is a water stopping layer, and 2-3 is an adsorption layer; fig. 5 is a schematic view of a retention layer structure provided by an embodiment of the invention, wherein the upper left drawing is a front view, the upper right drawing is a partial enlarged front view, the lower left drawing is a side view, and the lower right drawing is a partial enlarged side view. In the invention, the chromatographic paper with a three-layer structure comprises a retention layer 2-1, a water stop layer 2-2 and an adsorption layer 2-3 which are sequentially contacted, wherein the retention layer 2-1 is positioned at the top of the chromatographic paper, the retention layer 2-1 is in a funnel array structure and is used for retaining a coloring agent in a coloring agent solution, and a colored surface (namely, a calibration color layer) is formed on the chromatographic paper; the water stopping layer 2-2 and the absorption layer 2-3 are used for intercepting the effluent of the retention layer 2-1, so as to prevent the solution from flowing out of the chromatographic paper and polluting detection instrument and equipment. In the invention, the side length of the funnel array in the retention layer 2-1 is 0.2-0.8 μm, specifically 0.45 μm, and the distance between two adjacent funnels is preferably 0.05-0.5 μm, specifically 0.2 μm; the thickness of the detention layer 2-1 is preferably 0.2-1 mm, and specifically can be 0.6 mm; the thickness of the water stopping layer 2-2 is preferably 0.1-0.5 mm, and specifically can be 0.25 mm; the thickness of the adsorption layer 2-3 is preferably 0.5-2 mm, and specifically 1 mm.
The invention also provides a preparation method of the calibration color card for the biochemical detector, which comprises the following steps:
a) preparing a mixed dyeing solution containing a red dyeing agent and a blue dyeing agent, dripping the mixed dyeing solution onto the surface of the chromatographic paper, and forming a calibration color layer on the surface of the chromatographic paper;
b) and (3) filling the chromatographic paper with the calibration color layer into the calibration area groove of the color card shell, and buckling the calibration area cover plate of the color card shell to obtain the calibration color card for the biochemical detector.
In the preparation method provided by the invention, a mixed dyeing solution containing a red dyeing agent and a blue dyeing agent is prepared. Wherein the red coloring agent comprises one or more of fuchsin, rose bengal, rhodamine B and fluorescein, and is preferably fluorescein; the blue coloring agent comprises one or more of indigo disulfonic acid sodium, methine blue, Prussian blue and bromophenol blue, and is preferably methine blue. In the invention, the mixed dyeing liquid is prepared by mixing a red dyeing agent solution and a blue dyeing agent solution; the concentration of the coloring agent in the red coloring agent solution is preferably 2-10 g/50mL, and specifically can be 2g/50mL, 3g/50mL, 4g/50mL, 5g/50mL, 6g/50mL, 7g/50mL, 8g/50mL, 9g/50mL or 10g/50 mL; the concentration of the coloring agent in the blue coloring agent solution is preferably 150-250 mg/50mL, and specifically can be 150mg/50mL, 160mg/50mL, 170mg/50mL, 180mg/50mL, 190mg/50mL, 200mg/50mL, 210mg/50mL, 220mg/50mL, 230mg/50mL, 240mg/50mL or 250mg/50 mL. In the present invention, the solvent used for preparing the red and blue colorant solutions is determined according to the selected colorant type, wherein when the red colorant is fluorescein, the solvent used for preparing the red colorant solution is preferably alcohol, and the concentration of the alcohol is preferably 75 vol%; when the blue colorant is selected from methylene blue, the solvent used to prepare the blue colorant solution is preferably pure water. In the present invention, since commercially available fluorescein generally has a low purity (generally about 90%), it is preferable to perform a purification process when preparing an alcoholic solution of fluorescein, specifically, the preparation process is as follows: mixing the fluorescein crude product with alcohol, and filtering to remove impurities to obtain an alcohol solution of the fluorescein; the filter membrane adopted for filtering and impurity removing is preferably a glass fiber membrane, and the size of the filter pores of the glass fiber membrane is preferably 200-600 μm, and particularly can be 400 μm. In the present invention, the mixing ratio of the red colorant solution and the blue colorant solution is not particularly limited, and the ratio of the amount of the blue colorant solution is higher as the reactivity of the calibration color chart is higher, depending on the reactivity of the calibration color chart to be prepared.
In the preparation method provided by the invention, after the mixed dyeing solution is obtained, the mixed dyeing solution is dripped on the surface of the chromatographic paper, and a colored surface (namely, a calibration color layer) is formed on the chromatographic paper. In the above three-layer chromatography paper, the specific process of dropping the staining solution and forming the colored surface is shown in fig. 6, and fig. 6 is a schematic diagram of the process of trapping the staining agent and forming the colored surface by the retention layer of the chromatography paper provided in the embodiment of the present invention. Referring to fig. 6, after the mixed staining solution is dripped on the surface of the chromatographic paper, a red staining agent and a blue staining agent in the staining solution are intercepted by a retention layer of the chromatographic paper due to large particle size, so that a colored surface is formed on the chromatographic paper; and the solvent in the dyeing liquid enters the water stopping layer and the adsorption layer through the trapping layer.
In the preparation method provided by the invention, when the mixed staining solution is dripped, a constraint tool for constraining the diffusion of the staining solution is preferably arranged on the chromatographic paper, as shown in fig. 7, fig. 7 is an operation schematic diagram of dripping the mixed staining solution on the chromatographic paper provided with the constraint tool, provided by the embodiment of the invention, wherein 2 is the chromatographic paper, 3 is the constraint tool, and 4 is the droplet dropping device. In the invention, in order to enable the dyeing liquid to be better diffused when dripping, the constraint tool preferably has a circular through hole with a small top and a big bottom, and the draft angle of the through hole is preferably 5-15 degrees, and specifically can be 5 degrees, 6 degrees, 7 degrees, 8 degrees, 9 degrees, 10 degrees, 11 degrees, 12 degrees, 13 degrees, 14 degrees or 15 degrees. In the embodiment provided by the present invention, the constraint tool shown in fig. 8 may be specifically adopted, and fig. 8 is a schematic structural diagram of the constraint tool provided by the embodiment of the present invention, wherein the middle is a top view, the left side is a cross-sectional view, the right side is a side view, and the upper side is a front view.
In the preparation method provided by the invention, when the restraint tool is arranged in the process of dropwise adding the mixed dyeing solution, preferably after the dropwise adding of the mixed dyeing solution is finished, the restraint tool is removed, so that the mixed dyeing solution can be freely diffused on the chromatographic paper for a period of time, and a colored surface with better appearance is formed on the chromatographic paper.
In the preparation method provided by the invention, after the dripping process is completed, the chromatographic paper dripped with the dyeing liquid is properly cut according to the specification and the size of the calibration area of the shell of the color card, then the chromatographic paper is put into the groove of the calibration area of the shell of the color card, and the cover plate of the calibration area of the shell of the color card is buckled, so that the calibration color card for the biochemical detector is obtained.
In the preparation method provided by the invention, after the calibration color card is prepared, the reactivity of the calibration color card is measured on a standard instrument so as to calibrate the reactivity value of the calibration color card.
In the preparation method provided by the invention, in order to improve the stability of the calibration color card, the prepared calibration color card is preferably baked. Wherein the baking temperature is 40-50 ℃, and specifically can be 45 ℃; the baking time is preferably 10-60 min, and specifically can be 30 min.
According to the invention, the coloring agents with red and blue colors and solid granularity close to hemoglobin are selected and uniformly deposited on the surface of the chromatographic paper, so that the color card paper without biological components is obtained; the surface appearance of the color card paper is similar to the surface appearance formed after the surface affinity chromatography of the saccharified blood sample on the surface of the chromatographic paper, and the color card paper can be made into a standard sample for calibrating the total reactivity of the saccharified reflecting system of the biochemical detector. The invention installs the color card paper developed and designed based on the technical conception into the color card shell, thereby assembling and obtaining the calibration color card without biological components, and the calibration color card can be used for calibrating a biochemical detector which adopts red and blue light to detect, such as a boric acid affinity chromatography glycosylated hemoglobin detector. Experimental results show that the reflecting surface of the calibration color chart provided by the invention is very close to the surface of a saccharified blood sample formed on chromatographic paper, and the reactivity of a saccharified reflecting system of a biochemical detector can be effectively calibrated.
For the sake of clarity, the following examples are given in detail.
Example 1
1) Selecting chromatographic paper:
according to the adapted biochemical detector, the chromatographic paper adopted by the embodiment is consistent with the chromatographic structure used for testing the real sample, so that errors between calibration and real testing caused by different height positions and the like are avoided; more specifically, the chromatography paper is of a three-layer structure (shown in figure 4), and comprises a retention layer 2-1, a water stopping layer 2-2 and an adsorption layer 2-3, wherein the thickness of the retention layer 2-1 is 0.6mm, the thickness of the water stopping layer 2-2 is 0.25mm, and the thickness of the adsorption layer 2-3 is 1 mm; the retention layer 2-1 is of a funnel array structure (shown in FIG. 5), the side length of the funnel array in the retention layer 2-1 is 0.45 μm, and the distance between two adjacent funnels is 0.2 μm.
2) Preparation of red stain solution:
the fluorescein is insoluble in water but slightly soluble in alcohol, and is easy to form a flocculent structure when dissolved by water, so that the fluorescein is dissolved by alcohol; the concentration control of alcohol is also important when dissolving, and the higher the concentration, the faster the alcohol volatilizes, and a middle thick and thin-edged surface layer is formed when titrating and diffusing on a chromatographic structure. In this example, multiple experiments prove that a good chromatographic surface can be prepared by dissolving fluorescein in 75 vol% of alcohol and adding a constraint tool.
3) Preparation of blue stain solution:
methylene blue is soluble in alcohol and slightly soluble in water; when dissolved in alcohol, the methylene blue will fall from the funnel structure of the chromatography paper to the lower layer when dropped, and the surface cannot be well formed, so water is selected as the dissolving agent.
4) And (3) selecting a constraint tool:
in this embodiment, the restraining tool is a ring with a bottom diameter of 5mm, and a through hole is subjected to drawing by 10 degrees, so that better diffusion is achieved during dripping, and the specific structure of the restraining tool is shown in fig. 8. In the embodiment, the aperture size of the constraint tool is slightly larger than the reflection surface (diameter of 3.3mm) of the calibration color card, so that a certain allowance is reserved during manufacturing, and subsequent manufacturing and installation are facilitated.
5) The actual manufacturing process of the calibration color card comprises the following steps:
weighing 6.0g +/-0.1 g of fluorescein powder, introducing the fluorescein powder into a 50mL centrifuge tube or beaker, adding 75 vol% alcohol to 50mL scales, uniformly stirring (namely adding the alcohol into the fluorescein powder, wherein the total volume in a container is 50mL), filtering for 3-5 times by using glass fiber with 400-micrometer filter holes, and removing impurity components in the fluorescein to obtain a fluorescein solution; weighing 200mg +/-1 mg of methylene blue powder, introducing the methylene blue powder into a 50mL centrifuge tube or a beaker, adding purified water to 50mL scale, and uniformly stirring to fully dissolve the methylene blue powder (namely, the total volume of the methylene blue powder and the purified water in a container is 50mL) to obtain a methylene blue solution; uniformly mixing a fluorescein solution and a methine blue solution according to a certain proportion according to the requirement of the reactivity, wherein the basic principle of the mixing operation is that 1mL of the fluorescein solution is used as a substrate, and the reactivity of the mixed solution is adjusted according to 200 mu L of the methine blue solution each time; slowly dropping the mixed solution onto chromatographic paper constrained by a tool, removing the constraint tool, allowing the solution to freely diffuse for 1-2 minutes, and forming a good surface by visual observation to obtain the calibration color card paper; cutting the redundant part of the color card paper, then loading the cut redundant part into the groove of the calibration area of the color card shell shown in the figure 2, and then pressing the cut redundant part by using the cover plate of the calibration area shown in the figure 3 to manufacture the calibration color card shown in the figure 1; the reactivity of the calibration color card is measured on a standard instrument, the calibration color card is sent into a 45 ℃ oven to be baked immediately after the required value is determined, and a uniform and long-time stable reflecting surface can be formed on the calibration color card after half an hour.
Example 2
Comparing the reflection surface of the calibration color chart prepared in example 1 with the reflection surface of the blood sample color chart, the result is shown in fig. 9, fig. 9 is a comparison graph of the reflection surface of the calibration color chart provided in the example of the present invention and the reflection surface of the blood sample color chart under 20 times microscope, in which the calibration color chart is prepared under the condition that the dosage of the methine blue solution is 200 μ L; the blood sample color chart was prepared by the sample preparation method described in the specification of the boric acid affinity chromatography glycated hemoglobin meter. As can be seen from FIG. 9, the similarity of the chromatographic surface formed by the calibration color chart prepared in example 1 and real blood is very high under 20 times microscope.
Example 3
Calibration of biochemical detectors
1) The reactivity referred to in this protocol is defined as:
wherein R isRed wineAnd RBlue (B)Reflectance for red and blue light, respectively; the reactivity accords with Kubelka-Munk law, is in linear relation with the proportion of glycated hemoglobin of a blood sample in total protein, and can be directly measured by an instrument according to an algorithm.
2) Calibrating a saccharification reflection system of a biochemical detector by adopting a linear scheme, wherein the biochemical detector is a boric acid affinity chromatography saccharification hemoglobin detector, and light sources adopted by the saccharification reflection detection system of the detector are a blue light LED with the central wavelength of 460nm and a red light LED with the central wavelength of 640 nm; the calibration results were analyzed as follows:
preparing calibration color cards with uniform reactivity intervals and covering the detection range of the instrument by different mixing ratios of fluorescein and methylene blue, and performing linear fitting on the reactivity result of the calibration color card measured by a biochemical detector (hereinafter referred to as a testing machine) and the reactivity of the calibration color card measured by a standard instrument (hereinafter referred to as a standard machine) to obtain linear fitting coefficients K and B, wherein the specific results are shown in Table 1 and FIG. 10:
TABLE 1 reactivity values measured on a tester and a standard machine respectively for calibrating color cards and calibration results thereof
As can be seen from table 1 and fig. 10, the calibration result of the tester is quite good, the calibration coefficient reaches 0.99999, and the reactivity of the calibrated tester is at the same level as that of the standard tester.
Example 4
The calibrated testing machine of example 3 was used for testing blood samples, and the coefficient of variation (CV%) of the test results of low, medium and high values were analyzed, respectively, and the results are shown in table 2:
TABLE 2 test results of low, medium and high sample values of the calibrated tester
In Table 2, "Low value", "median" and "high value" refer to the concentration value of glycated hemoglobin in a blood sample; for example, "4.4" represents a blood sample having a glycated hemoglobin concentration of 4.4%.
As can be seen from Table 2, the instrument calibrated by the calibration color chart has high test precision, and meets the requirement that the CV% of the row standard requirement is not higher than 8%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A calibration color chart for a biochemical detector, comprising: the color card comprises a color card shell and a calibration color card paper arranged in a calibration area of the color card shell;
the calibration color card paper comprises chromatography paper and a calibration color layer arranged on the surface of the chromatography paper, wherein the calibration color layer contains a red coloring agent and a blue coloring agent;
the red coloring agent comprises one or more of fuchsin, rose bengal, rhodamine B and fluorescein;
the blue coloring agent comprises one or more of indigo disulfonic acid sodium, methine blue, Prussian blue and bromophenol blue.
2. The calibration color chart of claim 1, wherein the red stain is fluorescein; the blue colorant is methine blue.
3. The calibration color card of claim 1, wherein the chromatography paper comprises a retention layer, a water stop layer and an adsorption layer in sequential contact, the retention layer being located on top of the chromatography paper.
4. A preparation method of a calibration color card for a biochemical detector comprises the following steps:
a) preparing a mixed dyeing solution containing a red dyeing agent and a blue dyeing agent, dripping the mixed dyeing solution onto the surface of the chromatographic paper, and forming a calibration color layer on the surface of the chromatographic paper;
the red coloring agent comprises one or more of fuchsin, rose bengal, rhodamine B and fluorescein; the blue coloring agent comprises one or more of indigo disulfonic acid sodium, methine blue, Prussian blue and bromophenol blue;
b) and (3) filling the chromatographic paper with the calibration color layer into the calibration area groove of the color card shell, and buckling the calibration area cover plate of the color card shell to obtain the calibration color card for the biochemical detector.
5. The preparation method according to claim 4, wherein in the step a), the mixed dyeing liquid is prepared by mixing a red dyeing solution and a blue dyeing solution;
the concentration of the coloring agent in the red coloring agent solution is 2-10 g/50 mL; the concentration of the coloring agent in the blue coloring agent solution is 150-250 mg/50 mL.
6. The method according to claim 5, wherein the red colorant solution is an alcohol solution of fluorescein; the blue colorant solution is an aqueous solution of methylene blue.
7. The method according to claim 6, wherein the alcoholic solution of fluorescein is prepared by the following steps:
and mixing the fluorescein crude product with alcohol, and filtering to remove impurities to obtain an alcohol solution of the fluorescein.
8. The method according to claim 5, wherein a mixing ratio of the red colorant solution and the blue colorant solution is determined according to a reactivity of a calibration color chart to be prepared; the higher the reactivity of the calibration color chart to be prepared, the higher the proportion of the blue colorant solution.
9. The preparation method according to claim 4, wherein in the step a), when the mixed staining solution is dripped, a restraining tool for restraining the diffusion of the staining solution is arranged on the chromatographic paper;
the restraint frock has big end down's circular through-hole, and the draft of through-hole is 5 ~ 15.
10. The method of claim 4, further comprising: baking the calibration color card obtained in the step b).
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