Triglyceride detection reagent, triglyceride detection test paper and preparation method of triglyceride detection test paper
阅读说明：本技术 一种甘油三酯的检测试剂、检测试纸及试纸制备方法 (Triglyceride detection reagent, triglyceride detection test paper and preparation method of triglyceride detection test paper ) 是由 左园 宗小林 石丹垚 甘建民 崔娜娜 茹振楠 于 2020-08-12 设计创作，主要内容包括：本发明是一种甘油三酯的检测试剂、检测试纸及试纸制备方法,检测试剂包括0.5-1.5KU/ml的脂蛋白脂肪酶、0.3-0.5KU/ml的黄递酶、0.32-0.94KU/ml的甘油脱氢酶、5-15mmol/L的辅酶Ⅰ和2-6mmol/L的显色物质,还包括质量分数为0.2%-1.0%的TritonX-100溶液、0.01-0.1mol/L的缓冲液；显色物质为氯化硝基四氮唑蓝、二甲基噻唑二苯基四唑嗅蓝、二氯靛酚钠、TNBT、碘硝基氯化四氮唑蓝中的一种,检测试纸包括反应底层、反应层、滤血层、毛细进样层和亲水层,检测试剂位于反应层上表面。本发明测试结果受到其他物质的干扰影响较小,试剂检测更为稳定。(The invention relates to a triglyceride detection reagent, a detection test paper and a test paper preparation method, wherein the detection reagent comprises 0.5-1.5KU/ml lipoprotein lipase, 0.3-0.5KU/ml diaphorase, 0.32-0.94KU/ml glycerol dehydrogenase, 5-15mmol/L coenzyme I and 2-6mmol/L chromogenic substance, and also comprises 0.2-1.0% TritonX-100 solution and 0.01-0.1mol/L buffer solution according to mass fraction; the chromogenic substance is one of nitrotetrazolium chloride, dimethylthiazolediphenyltetrazole olfactory blue, sodium dichlorophenolate, TNBT and iodonitrotetrazolium chloride, the detection test paper comprises a reaction bottom layer, a reaction layer, a blood filtering layer, a capillary sample injection layer and a hydrophilic layer, and the detection reagent is positioned on the upper surface of the reaction layer. The test result of the invention is less influenced by the interference of other substances, and the reagent detection is more stable.)
1. The triglyceride detection reagent is characterized by comprising 0.5-1.5KU/ml lipoprotein lipase, 0.3-0.5KU/ml diaphorase, 0.32-0.94KU/ml glycerol dehydrogenase, 5-15mmol/L coenzyme I and 2-6mmol/L chromogenic substance, and also comprising 0.2-1.0% by mass of TritonX-100 solution and 0.01-0.1mol/L buffer solution;
the chromogenic substance is one of nitrotetrazolium chloride blue, dimethylthiazolediphenyltetrazole olfactory blue, dichloroindophenol sodium, TNBT and iodonitrotetrazolium chloride blue;
the buffer solution is one of Tris-HCl buffer solution, CAPS buffer solution, glycine-sodium hydroxide buffer solution, boric acid-borax buffer solution, borax-sodium hydroxide buffer solution and potassium carbonate-sodium bicarbonate buffer solution;
the pH of the buffer solution is 8.0-11.0.
2. The test paper added with the triglyceride detection reagent of claim 1 is characterized by comprising a reaction bottom layer (1), a reaction layer (2), a blood filter layer (3), a capillary sample injection layer (4) and a hydrophilic layer (5) which are sequentially stacked from bottom to top; the reagent for detecting triglyceride is coated on the upper surface of the reaction layer (2).
3. The test strip with the triglyceride detection reagent added thereto according to claim 2, wherein the reaction substrate (1) is a long sheet-shaped thin plate, and the reaction substrate (1) is provided with the reflection detection hole (6).
4. The test strip with the triglyceride detection reagent added thereto according to claim 3, wherein the reaction substrate (1) is made of any one of polyvinyl chloride, polyesteramide, polycarbonate and polyester, and the thickness of the reaction substrate (1) is 0.2 mm.
5. The test strip with the triglyceride detection reagent added thereto according to claim 4, wherein the reaction layer (2) is a long strip-shaped sheet-like film, the width of the reaction layer (2) is the same as the width of the reaction bottom layer (1) and the length of the reaction layer is smaller than the length of the reaction bottom layer (1); the reaction layer (2) is provided with a reaction area (7), the reaction area (7) is an annular groove, and the center of the annular groove of the reaction area (7) is arranged corresponding to the center of the reflection detection hole (6).
6. The test strip with the triglyceride detection reagent added thereto according to claim 5, wherein the blood filter layer (3) is a long strip-shaped sheet film, the size of the blood filter layer (3) is consistent with the size of the reaction layer (2), a blood filter area (8) is arranged on the blood filter layer (3), the blood filter area (8) is an annular groove, and the annular groove of the blood filter area (8) corresponds to the annular groove of the reaction area (7).
7. The test paper added with the triglyceride detection reagent is characterized in that two double-sided adhesive tapes (9) are arranged on the upper surface of the capillary sample injection layer (4) along the length direction, a channel is formed between the two double-sided adhesive tapes (9) on the upper surface of the capillary sample injection layer (4), diffusion holes (10) are arranged in the channel of the capillary sample injection layer (4), the diameter of each diffusion hole (10) is the same as that of the reflection detection hole (6), and the diffusion holes (10) on the channel of the capillary sample injection layer (4) and the blood filtration area (8) are concentrically arranged.
8. The test strip with the triglyceride detection reagent added thereto according to claim 7, wherein the hydrophilic layer (5) is a long strip-shaped sheet-shaped film, the length of the hydrophilic layer (5) is the same as the length of the double-sided tape (9) and the width is the same as the interval width of the two double-sided tapes (9), and the hydrophilic layer (5) completely covers the two double-sided tapes (9) and the channel.
9. The test strip with the triglyceride detection reagent added thereto according to claim 8, wherein the hydrophilic layer (5) is made of polyester or polyvinylpyrrolidone.
10. The method for preparing the test paper with the triglyceride detection reagent added according to claim 9, which comprises the following steps:
s1, selecting a reaction bottom layer (1), and processing a reflection detection hole (6) on the reaction bottom layer (1);
s2, adhering the reaction layer (2) provided with the reaction zone (7) to the reaction bottom layer (1) through double-sided adhesive, and ensuring that the reaction zone (7) is aligned with the center of the reflection detection hole (6);
s3, preparing a triglyceride detection reagent according to a ratio, dropwise adding the prepared triglyceride detection reagent into a reaction area (7) of a reaction layer (2) by using a liquid dropping machine, wherein the sample adding amount of each hole is 1 mu l, and drying the liquid after dropwise adding, wherein the drying temperature is 37 ℃ and the drying time is 20 min;
s4, attaching the blood filtering layer (3) on the reaction layer (2) to ensure that the blood filtering area (8) on the blood filtering layer (3) is aligned with the reaction area (7);
s5, attaching the capillary sample injection layer (4) on the blood filtration layer (3), attaching two long double-sided adhesive tapes (9) on the upper surface of the capillary sample injection layer (4), forming a long and narrow channel between the two long double-sided adhesive tapes (9), and ensuring that a diffusion hole (10) on the channel of the capillary sample injection layer (4) and the blood filtration region (8) are concentrically arranged
S6, adhering the hydrophilic layer (5) and the double-sided tape (9) to enable the hydrophilic layer (5) to cover the whole channel and the double-sided tape (9);
and S7, finally cutting to obtain the triglyceride detection test paper.
Technical Field
The invention relates to the technical field of triglyceride detection, in particular to a triglyceride detection reagent, triglyceride detection test paper and a preparation method of the triglyceride detection test paper.
Background
The currently widely used dry chemical test method for triglyceride is to use lipoprotein esterase, glycerol kinase, glycerol phosphate oxidase to couple Trinder reaction. However, the redox color developing agent in the system is very unstable when exposed to air or light, and if the redox color developing agent is not stored properly, the redox color developing agent is easily oxidized in advance to generate color change before the product is used, so that the product is invalid or the detection result is inaccurate.
In the existing solution, for example, in patent CN105295441B, a developer stabilizer, a reducing agent, a chelating agent, an auxiliary stabilizer and other various auxiliaries are added into a formula, so that the developer keeps better color development capability; patent CN102128919B also adopts two color stabilizers, one of which has stronger reducing power than the developer, and the other has reducing power between the developer and the first color stabilizer, but the addition of the reducing stabilizer in the two patents has certain interference effect on the redox reaction of the developer, and has great influence on the detection result.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a triglyceride detection reagent, a triglyceride detection test paper and a preparation method of the triglyceride detection test paper.
In order to achieve the purpose, the invention adopts the following technical scheme:
a triglyceride detection reagent comprises 0.5-1.5KU/ml lipoprotein lipase, 0.3-0.5KU/ml diaphorase, 0.32-0.94KU/ml glycerol dehydrogenase, 5-15mmol/L coenzyme I and 2-6mmol/L chromogenic substance, and also comprises TritonX-100 solution with mass fraction of 0.2-1.0% and 0.01-0.1mol/L buffer solution;
the chromogenic substance is one of nitrotetrazolium chloride blue, dimethylthiazolediphenyltetrazole olfactory blue, dichloroindophenol sodium, TNBT and iodonitrotetrazolium chloride blue;
the buffer solution is one of Tris-HCl buffer solution, CAPS buffer solution, glycine-sodium hydroxide buffer solution, boric acid-borax buffer solution, borax-sodium hydroxide buffer solution and potassium carbonate-sodium bicarbonate buffer solution;
the pH of the buffer solution is 8.0-11.0.
The test paper added with the triglyceride detection reagent comprises a reaction bottom layer, a reaction layer, a blood filtration layer, a capillary sample injection layer and a hydrophilic layer which are sequentially stacked from bottom to top; the reagent for detecting triglyceride is coated on the upper surface of the reaction layer.
The reaction bottom layer is a strip sheet-shaped thin plate, and a reflection detection hole is formed in the reaction bottom layer.
The reaction bottom layer is made of any one of polyvinyl chloride, polyesteramide, polycarbonate and polyester, and the thickness of the reaction bottom layer is 0.2 mm.
The reaction layer is a strip sheet-shaped film, the width of the reaction layer is consistent with that of the reaction bottom layer, and the length of the reaction layer is smaller than that of the reaction bottom layer; the reaction layer is provided with a reaction zone, the reaction zone is an annular groove, and the center of the annular groove of the reaction zone corresponds to the center of the reflection detection hole.
The blood filtering layer is a strip-shaped sheet film, the size of the blood filtering layer is consistent with that of the reaction layer, a blood filtering area is arranged on the blood filtering layer and is an annular groove, and the annular groove of the blood filtering area corresponds to the annular groove of the reaction area.
The capillary advances kind layer upper surface and is equipped with two double-sided tape along length direction, capillary advances kind layer upper surface and forms the channel between two double-sided tape, be equipped with the diffusion hole in the channel on capillary advances kind layer, the diameter in diffusion hole is the same with the diameter of reflection inspection hole, and diffusion hole and the concentric setting of blood filtration district on the capillary advances kind layer channel.
Hydrophilic layer is rectangular slice film, hydrophilic layer length is the same with two-sided tape's length and width and two-sided tape's interval width the same, hydrophilic layer covers completely on two-sided tape and channel.
The hydrophilic layer is made of polyester and polyvinylpyrrolidone.
The preparation method of the test paper added with the triglyceride detection reagent comprises the following specific steps:
s1, selecting a reaction bottom layer, and processing a reflection detection hole on the reaction bottom layer;
s2, adhering the reaction layer with the reaction zone on the reaction bottom layer through double-sided adhesive, and ensuring the reaction zone to be aligned with the center of the reflection detection hole;
s3, preparing a triglyceride detection reagent according to a ratio, dropwise adding the prepared triglyceride detection reagent into a reaction area of a reaction layer by using a liquid dropping machine, wherein the sample adding amount of each hole is 1 mu l, and drying the liquid after dropwise adding, wherein the drying temperature is 37 ℃ and the drying time is 20 min;
s4, attaching a blood filtering layer on the reaction layer to ensure that a blood filtering area on the blood filtering layer is aligned with the reaction area;
s5, attaching a capillary sample injection layer on the blood filtration layer, attaching two long double-sided adhesive tapes on the upper surface of the capillary sample injection layer, and forming a long and narrow channel between the two long double-sided adhesive tapes to ensure that a diffusion hole on the channel of the capillary sample injection layer and the blood filtration region are concentrically arranged;
s6, adhering the hydrophilic layer and the double-sided tape to enable the hydrophilic layer to cover the whole channel and the double-sided tape;
and S7, finally cutting to obtain the triglyceride detection test paper.
The invention has the beneficial effects that: the dry chemical detection adopts a colorimetric test route of glycerol dehydrogenase, the adopted chromogenic substance catalyzes and generates an insoluble blue product, and the intensity of the generated chromogenic is related to the concentration of triglyceride and is different from the commonly adopted lipoprotein esterase, glycerol kinase and glycerophosphate oxidase coupled Trinder reaction. The color developing substance adopted by the invention is a stable color developing agent, so that a plurality of protective agents are not required to be added, the test result is less influenced by the interference of other substances, and the test paper is more stable in detection.
Drawings
FIG. 1 is a schematic structural diagram of the test paper of the present invention;
FIG. 2 is a standard graph of triglyceride concentration versus Δ AD value for comparative and example samples of test triglyceride serum;
in the figure: 1-a reaction bottom layer; 2-a reaction layer; 3-a blood filtration layer; 4-capillary sample injection layer; 5-a hydrophilic layer; 6-reflection detection hole; 7-a reaction zone; 8-a blood filtration zone; 9-double-sided tape; 10-diffusion holes;
the following detailed description will be made in conjunction with embodiments of the present invention with reference to the accompanying drawings.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings:
a triglyceride detection reagent comprises 0.5-1.5KU/ml lipoprotein lipase, 0.3-0.5KU/ml diaphorase, 0.32-0.94KU/ml glycerol dehydrogenase, 5-15mmol/L coenzyme I and 2-6mmol/L chromogenic substance, and also comprises TritonX-100 solution with mass fraction of 0.2-1.0% and 0.01-0.1mol/L buffer solution;
the chromogenic substance is one of nitrotetrazolium chloride blue, dimethylthiazolediphenyltetrazole olfactory blue, dichloroindophenol sodium, TNBT and iodonitrotetrazolium chloride blue;
the buffer solution is one of Tris-HCl buffer solution, CAPS buffer solution, glycine-sodium hydroxide buffer solution, boric acid-borax buffer solution, borax-sodium hydroxide buffer solution and potassium carbonate-sodium bicarbonate buffer solution;
the pH of the buffer solution is 8.0-11.0.
As shown in fig. 1, the test paper added with the triglyceride detection reagent comprises a
The
The
The
The blood filtering layer 3 is a strip-shaped sheet film, the size of the blood filtering layer 3 is consistent with that of the
The capillary advances
The
The preparation method of the test paper added with the triglyceride detection reagent comprises the following specific steps:
s1, selecting a
s2, adhering the
s3, preparing a triglyceride detection reagent according to a ratio, dropwise adding the prepared triglyceride detection reagent into the
s4, attaching the blood filtering layer 3 on the
s5, attaching the capillary
s6, adhering the
and S7, finally cutting to obtain the triglyceride detection test paper.
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