CN116121336B - Blood fat three-item test strip and preparation method thereof - Google Patents

Abstract

The application provides a blood fat three-item test strip and a preparation method thereof, and relates to the field of medical detection. The blood fat three-item test strip comprises a transparent substrate, a color developing layer, a reaction layer, a blood filtering layer, a diffusion layer, a siphon groove layer and a hydrophilic film layer which are sequentially laminated from bottom to top; the color development layer, the reaction layer, the blood filtering layer and the diffusion layer have porous structures and are formed by the reagents in the corresponding layers. The preparation method of the blood fat three-item test strip comprises the following steps: sequentially arranging reagents of a color developing layer, a reaction layer, a blood filtering layer and a diffusion layer on a transparent substrate, and drying and forming to obtain corresponding layers; and (3) arranging a siphon groove layer and a hydrophilic film layer, and performing post-treatment to obtain the blood fat three-item test strip. The three blood lipid test strips effectively solve the problems that the prior dry chemical test strip film materials are overlapped layer by layer, the retention rate of blood samples is high, the production process involves complex processes such as liquid dropping or film soaking, assembly, shell clamping and the like, and the cost is high, the operation is complex, the blood consumption is large and the like.

Description

Blood fat three-item test strip and preparation method thereof

Technical Field

The application relates to the field of medical detection, in particular to a blood fat three-item test strip and a preparation method thereof.

Background

Cardiovascular disease is a common disease that severely threatens the health of humans, particularly the elderly over 50 years old. Hyperlipidemia is one of the direct causes of cardiovascular disease, and elevated levels of TC, TG and LDL and reduced levels of HDL-C are the major lipid risk factors for the development and progression of atherosclerosis, as well as risk factors for the induction of coronary heart disease. Therefore, the periodic detection of TC, TG, HDL-C, LDL-C has important significance for preventing and treating cardiovascular and cerebrovascular diseases.

The portable blood lipid detection product is simple to operate, quick and accurate in measurement, can monitor the blood lipid level of a patient at any time, and provides an early warning for potential patients with hyperlipidemia, thereby providing scientific basis for healthy life and reasonable medication. The dry chemical blood lipid combined test strip is formed by combining membrane materials such as a substrate, a reaction membrane, a background removing membrane, a blood filtering membrane, a diffusion membrane and the like in a gluing or welding mode, and is externally fixed by a clamping shell. The sample passes through the diffusion membrane and then rapidly and uniformly diffuses to reach the blood filtering membrane, and red blood cells and the like are trapped by the filtration of the blood filtering membrane. The effective serum reaches the background and reaction removal membranes. Different reagents are fixed on the reaction membrane, so that the test of different indexes can be realized. The technology is mature at present.

However, as the test strip membrane materials are overlapped layer by layer, the retention rate of the blood sample is higher; the raw materials such as the used membrane material are mostly imported, and the cost is high. The test strip production process involves the processes of liquid dispensing or film soaking, assembly, shell clamping and the like, and is relatively complex. In the test process, a capillary is required to collect a sample, and the operation is complex. The blood consumption is generally high, and is 30-40 mu L.

In addition, the film materials used in the existing test strips are generally derived from different film material manufacturers, and the difference between material batches is slightly larger due to the difference of transportation and storage conditions, in addition, the lamination degree between film layers cannot be controlled in the assembly and cutting processes, the difference of humidity in the assembly environment can cause the difference of electrostatic actions between film layers, and the above reasons can cause the larger difference between batches of the commercial test strips.

Disclosure of Invention

The application aims to provide a blood fat three-item test strip and a preparation method thereof, so as to solve the problems.

In order to achieve the above purpose, the present application adopts the following technical scheme:

a blood fat three-item test strip comprises a transparent substrate, a color developing layer, a reaction layer, a blood filtering layer, a diffusion layer, a siphon groove layer and a hydrophilic film layer which are sequentially laminated from bottom to top;

the chromogenic layer comprises an independent cholesterol chromogenic region, a high-density lipoprotein chromogenic region and a triglyceride chromogenic region; the cholesterol color development zone, the high density lipoprotein color development zone and the triglyceride color development zone are formed by sequentially arranging color development zone film forming agent, color development agent and color development zone isolating agent;

the reaction layer comprises an independent cholesterol reaction zone, a high-density lipoprotein reaction zone and a triglyceride reaction zone; the cholesterol reaction zone, the high-density lipoprotein reaction zone and the triglyceride reaction zone are formed by sequentially arranging a reaction zone film forming agent, a reaction reagent and a reaction zone isolating agent;

the cholesterol color development zone is arranged corresponding to the cholesterol reaction zone, the high-density lipoprotein color development zone is arranged corresponding to the high-density lipoprotein reaction zone, and the triglyceride color development zone is arranged corresponding to the triglyceride reaction zone;

the color developing layer, the reaction layer, the blood filtering layer and the diffusion layer have porous structures and are formed by the reagents in the corresponding layers.

The transparent substrate is used as a supporting layer, and the surface of the transparent substrate can be physically divided to form three independent areas corresponding to the color development layer and the reaction layer. The physical dividing mode includes but is not limited to glue dividing line, hollowed dividing and the like.

Preferably, the chromogenic region film former comprises cellulose acetate 20-60g/m ³ 40-400g/m of first white reflecting material ³ 40-400g/m of silicon dioxide ³ 40-400g/m diatomite ³ Microporous material 40-400g/m, natural and/or synthetic ³ ；

The first white reflecting material is titanium dioxide and/or barium sulfate.

Alternatively, the amount of cellulose acetate in the chromogenic region film former may be 20g/m ³ 、30 g/m ³ 、40 g/m ³ 、50 g/m ³ 、60 g/m ³ Or 20-60g/m ³ Any value in between; the first white reflective material may be used in an amount of 40 g/m ³ 、100 g/m ³ 、200 g/m ³ 、300 g/m ³ 、400 g/m ³ Or 40-400g/m ³ Any value in between; the amount of silica may be 40 g/m ³ 、100 g/m ³ 、200 g/m ³ 、300 g/m ³ 、400 g/m ³ Or 40-400g/m ³ Any value in between; the diatomite dosage can be 40 g/m ³ 、100 g/m ³ 、200 g/m ³ 、300 g/m ³ 、400 g/m ³ Or 40-400g/m ³ Any value in between; the natural and/or synthetic microporous material may be used in an amount of 40 g/m ³ 、100 g/m ³ 、200 g/m ³ 、300 g/m ³ 、400 g/m ³ Or 40-400g/m ³ Any value in between;

synthesis of titanium dioxide white reflecting material:

commercially available titanium dioxide particles were dispersed in ethanol, and aqueous ammonia (28 wt%) was added. TBOT was then added and the reaction was stirred. And then centrifuging, washing with water and ethanol, and drying the obtained powder. Finally, the resulting sample is freed from organic species and the degree of crystallization is increased.

Screening mesoporous TiO with different specifications and different concentrations according to different functional area requirements ₂ As microporous materials, film forming agents in different regions are formulated. The components, particle size and color of the white reflecting material in the film forming reagent are one of the main factors influencing the uniformity of test results of the test paper.

The barium sulfate is commercially available common barium sulfate.

Preferably, the color development zone separating agent comprises gelatin, xanthan gum, agar and its derivatives, and the content is 5-50 g/m ³ ；

Alternatively, the color-developing zone-separating agent may be present in an amount of 5 g/m ³ 、10 g/m ³ 、15 g/m ³ 、20 g/m ³ 、25 g/m ³ 、30 g/m ³ 、35 g/m ³ 、40 g/m ³ 、45 g/m ³ 、50 g/m ³ Or 5-50 g/m ³ Any value in between;

the reaction zone separating agent comprises gelatin, xanthan gum, agar and its derivatives, and the content is 5-50 g/m ³ 。

Alternatively, the reaction zone separating agent may be present in an amount of 5 g/m ³ 、10 g/m ³ 、15 g/m ³ 、20 g/m ³ 、25 g/m ³ 、30 g/m ³ 、35 g/m ³ 、40 g/m ³ 、45 g/m ³ 、50 g/m ³ Or 5-50 g/m ³ Any value in between.

Preferably, each of the cholesterol color development zone, the high density lipoprotein color development zone and the color development agent of the triglyceride color development zone independently comprises a first color development reagent of 0.5-5mM, a first buffer of 50-1000mM, a first surfactant of 0.1-2g/L, a first protecting agent of 0.5-5mM and a first preservative of 0.1-5.0 g/L;

alternatively, the amount of the first color-developing reagent in the color-developing reagent may be any value between 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, or 0.5-5 mM; the amount of the first buffer may be 50mM, 100mM, 200mM, 300mM, 400mM, 500mM, 600mM, 700mM, 800mM, 900mM, 1000mM or any value between 50 and 1000 mM; the amount of the first surfactant may be 0.1g/L, 0.5g/L, 1g/L, 1.5g/L, 2g/L, or any value between 0.1 and 2 g/L; the amount of the first protecting agent may be any value between 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, or 0.5-5 mM; the first preservative may be used in an amount of 0.1g/L, 0.5g/L, 1g/L, 1.5g/L, 2g/L, 2.5g/L, 3g/L, 3.5g/L, 4g/L, 4.5g/L, 5g/L, or any value between 0.1 and 5.0g/L;

the first color reagent comprises one or more of N- (2-hydroxy-3-sulfopropyl) -3', 5-dimethoxy aniline sodium salt, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and 4-aminoantipyrine;

the buffer solution comprises any one of phosphate buffer solution, tris-HCl buffer solution or 3- (N-morpholinyl) propane sulfonic acid buffer solution, and the pH value is 6.5-7.5;

alternatively, the pH of the buffer may be any value between 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5 or 6.5-7.5;

the first surfactant comprises one or more of laureth-35 (Brij-35), polyethylene glycol octadecyl ether, sodium dodecyl benzene sulfonate, tween-20, tween-40, tween-60, triton-100 and triton-405;

the first protective agent comprises one or more of Bovine Serum Albumin (BSA), trehalose, fish gelatin, mannitol and polyvinyl alcohol;

the first preservative comprises one or more of sodium azide, sodium benzoate, proclin 300, krovin-100.

Preferably, the reaction zone film former comprises cellulose acetate 10-50g/m ³ Second white reflecting material 20-200g/m ³ Second surfactant 2-10 g/m ³ ；

Alternatively, the amount of cellulose acetate in the reaction zone film former may be 10g/m ³ 、20 g/m ³ 、30 g/m ³ 、40 g/m ³ 、50 g/m ³ Or 10-50g/m ³ Any value in between; the second white reflective material can be used in an amount of 20g/m ³ 、50 g/m ³ 、100 g/m ³ 、150 g/m ³ 、200 g/m ³ Or 20-200g/m ³ Any value in between; the second surfactant may be used in an amount of 2 g/m ³ 、5 g/m ³ 、10 g/m ³ Or 2-10 g/m ³ Any value in between;

the second white reflecting material is titanium dioxide and/or barium sulfate;

the second surfactant comprises one or more of laureth-35, polyethylene glycol octadecyl ether, sodium dodecyl benzene sulfonate, tween-20, tween-40, tween-60, triton-100, triton-405 and OP-10.

Preferably, the reaction reagent of the cholesterol reaction zone comprises 10-200KU/L of cholesterol oxidase, 10-100KU/L of cholesterol esterase, 50-500mM of second buffer solution, 0.5-10mM of second protective agent, 0.1-5.0g/L of third surfactant and 0.5-5.0g/L of second preservative, the reaction reagent of the high-density lipoprotein reaction zone comprises 10-100KU/L of cholesterol oxidase, 10-50KU/L of cholesterol esterase, 50-500mM of third buffer solution, 0.5-10mM of third protective agent, 1-5g/L of precipitant, 0.1-5.0g/L of fourth surfactant and 0.5-5.0g/L of third preservative, and the reaction reagent of the triglyceride reaction zone comprises 50-200 KU/L of glycerol kinase, 50-500mM of fourth buffer solution, 0.5-5mM of fourth protective agent, 0.1-5 g/L of fifth surfactant and 0.0.1-5 g/L of fourth preservative;

alternatively, the cholesterol oxidase may be used in an amount of 10KU/L, 20KU/L, 50KU/L, 100KU/L, 150KU/L, 200KU/L or any value between 10 and 200KU/L in the reaction reagent in the cholesterol reaction region; the cholesterol esterase may be used in an amount of any of 10KU/L, 20KU/L, 50KU/L, 100KU/L, or 10-100 KU/L; the amount of the second buffer may be 50mM, 100mM, 200mM, 300mM, 400mM, 500mM or any value between 50 and 500 mM; the second protective agent may be used in an amount of any of 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, or between 0.5 and 10 mM; the amount of the third surfactant may be or be any value between 0.1 and 5.0g/L; the second preservative may be used in an amount of any of 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, or 0.5-5.0g/L; the cholesterol oxidase may be used in an amount of 10KU/L, 50KU/L, 100KU/L or 10-100KU/L in the reagent layer of the high density lipoprotein reaction zone; the cholesterol esterase may be used in an amount of 10KU/L, 20KU/L, 30KU/L, 40KU/L, 50KU/L or any value between 10 and 50 KU/L; the amount of the third buffer may be 50mM, 100mM, 200mM, 300mM, 400mM, 500mM or any value between 50 and 500 mM; the third protecting agent may be used in an amount of any of 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, or between 0.5 and 10 mM; the amount of precipitant may be 1g/L, 2g/L, 3g/L, 4g/L, 5g/L or any value between 1 and 5 g/L; the fourth surfactant may be used in an amount of any of 0.1g/L, 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, or 0.1-5.0 g/L; the third preservative may be used in an amount of any of 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, or 0.5-5.0g/L; the amount of glycerol kinase in the reagent in the triglyceride reaction zone may be any value between 50KU/L, 100KU/L, 150KU/L, 200KU/L or 50-200 KU/L; the fourth buffer may be used in an amount of any of 50mM, 100mM, 200mM, 300mM, 400mM, 500mM, or between 50 and 500 mM; the fourth protectant may be used in an amount of any of 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, or between 0.5 and 5 mM; the fifth surfactant may be used in an amount of any of 0.1g/L, 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, or 0.1-5.0 g/L; the fourth preservative may be used in an amount of any of 0.5g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, or 0.5-5.0g/L;

the second buffer solution, the third buffer solution and the fourth buffer solution respectively and independently comprise any one of a phosphate buffer solution, a Tris-HCl buffer solution or a 3- (N-morpholinyl) propane sulfonic acid buffer solution, the pH value of the second buffer solution is 6.5-7.0, the pH value of the third buffer solution is 6.5-7.0, and the pH value of the fourth buffer solution is 7.0-7.5;

alternatively, the pH of the second buffer may be any value between 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, or 6.5-7.0, the pH of the third buffer may be any value between 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, or 6.5-7.0, and the pH of the fourth buffer may be any value between 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.0-7.5;

the second protective agent and the third protective agent respectively independently comprise one or more of bovine serum albumin, trehalose and glycerol, and the fourth protective agent comprises one or more of serum albumin, trehalose, polyvinylpyrrolidone and polyvinyl alcohol;

the third surfactant, the fourth surfactant and the fifth surfactant independently comprise one or more of laureth-35, polyethylene glycol octadecyl ether, sodium dodecyl benzene sulfonate, tween-20, tween-40, triton 405 and OP-10;

the precipitant comprises one or more of phosphotungstic acid, dextran sulfate and polyethylene glycol;

the second preservative, the third preservative and the fourth preservative each independently comprise one or more of sodium azide, sodium benzoate, proclin 300 and Krovin-100.

Preferably, the blood filtering layer comprises cellulose acetate 2-20g/m ³ 40-400g/m of third white reflecting material ³ 40-400g/m diatomite ³ Natural and/or synthetic microporous materials 20-200g/m ³ Lectin 1-10g/m ³ ；

Optionally, the dosage of cellulose acetate in the blood filtering layer is 2 g/m ³ 、5 g/m ³ 、10 g/m ³ 、15 g/m ³ 、20 g/m ³ Or 2-20g/m ³ Any value in between; the third white reflective material can be used in an amount of 40 g/m ³ 、50 g/m ³ 、100 g/m ³ 、150 g/m ³ 、200 g/m ³ 、250 g/m ³ 、300 g/m ³ 、350 g/m ³ 、400 g/m ³ Or 40-400g/m ³ Any value in between; the diatomite dosage can be 40 g/m ³ 、50 g/m ³ 、100 g/m ³ 、150 g/m ³ 、200 g/m ³ 、250 g/m ³ 、300 g/m ³ 、350 g/m ³ 、400 g/m ³ Or 40-400g/m ³ Any value in between; the natural and/or synthetic microporous material may be used in an amount of 20g/m ³ 、50 g/m ³ 、100 g/m ³ 、150 g/m ³ 、200 g/m ³ Or 20-200g/m ³ Any value in between; lectin may be used in an amount of 1 g/m ³ 、2 g/m ³ 、3 g/m ³ 、4 g/m ³ 、5 g/m ³ 、6 g/m ³ 、7 g/m ³ 、8 g/m ³ 、9 g/m ³ 、10 g/m ³ Or 1-10g/m ³ Any value in between;

the third white reflecting material is titanium dioxide and/or barium sulfate, and the lectin comprises one or more of Canavalia gladiata lectin A, wheat germ element and soybean lectin; natural microporous materials include microporous cellulose and synthetic microporous materials include resins and/or silica.

Preferably, the diffusion layer comprises cellulose acetate 20-60g/m ³ Fourth white reflecting material 20-200g/m ³ Diatomite 100-500g/m ³ Microporous material of 100-500g/m, natural and/or artificial synthesis ³ Sixth surfactant 2-20g/m ³ ；

Alternatively, the amount of cellulose acetate in the diffusion layer may be 20g/m ³ 、30 g/m ³ 、40 g/m ³ 、50 g/m ³ 、60 g/m ³ Or 20-60g/m ³ Any value in between; the dosage of the fourth white reflecting material can be 20g/m ³ 、40 g/m ³ 、50 g/m ³ 、100 g/m ³ 、150 g/m ³ 、200 g/m ³ Or 20-200g/m ³ Any value in between; the dosage of the diatomite can be100g/m ³ 、200 g/m ³ 、300 g/m ³ 、400 g/m ³ 、500 g/m ³ Or 100-500g/m ³ Any value in between; the natural and/or synthetic microporous material may be used in an amount of 100g/m ³ 、200 g/m ³ 、300 g/m ³ 、400 g/m ³ 、500 g/m ³ Or 100-500g/m ³ Any value in between; the sixth surfactant may be used in an amount of 2 g/m ³ 、5 g/m ³ 、10 g/m ³ 、15 g/m ³ 、20 g/m ³ Or 2-20g/m ³ Any value in between;

the fourth white reflecting material is titanium dioxide and/or barium sulfate, the natural microporous material comprises microporous cellulose, and the artificially synthesized microporous material comprises resin and/or silicon dioxide; the sixth surfactant comprises one or more of laureth-35, polyethylene glycol octadecyl ether, sodium dodecyl benzene sulfonate, tween-20, tween-40, triton 405 and OP-10.

The mesoporous silica with good biocompatibility and different particle diameters is synthesized.

By adjusting the concentration of the reaction substances and the reaction conditions, microporous materials with good hydrophilicity and different particle diameters are synthesized.

The specific synthesis steps are as follows:

synthesis of silica:

measuring ethanol and ammonia water (28 wt%) and uniformly stirring, then adding TEOS (tetraethyl orthosilicate) to make reaction, and regulating quantity of TEOS, stirring temperature and speed so as to synthesize SiO with different grain sizes and forms ₂ 。

Mesoporous SiO with different concentrations ₂ As microporous materials, film forming agents in different regions are formulated. The microporous material component in the film forming reagent is one of the main factors affecting the blood filtering and diffusion properties of the test paper.

Preferably, the material of the transparent substrate includes any one of polycarbonate and PET, PVC, PE.

The application also provides a preparation method of the blood fat three-item test strip, which comprises the following steps:

sequentially arranging reagents of the color development layer, the reaction layer, the blood filtering layer and the diffusion layer on the transparent substrate, and drying and forming to obtain corresponding layers;

and setting the siphon groove layer and the hydrophilic film layer, and performing post-treatment to obtain the blood fat three-item test strip.

The transparent substrate is sequentially arranged, and the method can be coating, spraying or printing.

Compared with the prior art, the beneficial effects of this application include:

the three test strips of blood fat that this application provided, chromogenic layer and reaction layer detect the region that corresponds with cholesterol, high density lipoprotein, triglyceride respectively, have realized the three targets of simultaneous detection blood fat, can effectively reduce blood sampling volume.

The color development layer, the reaction layer, the blood filtering layer and the diffusion layer are formed by the reagents in the corresponding layers, so that the existing method of using a membrane material as a loading base material and loading corresponding detection reagents is abandoned, and the problems of higher blood sample retention rate, high membrane material cost, complex production and assembly and the like are solved; the reagent is molded to form the film to obtain each layer, expensive reagents are not needed to be selected, the cost is low, the stability of the film forming process is good, and the uniformity of each layer of the test strip after film forming is high. The problems that the traditional test strip is formed by physically superposing a plurality of layers of membrane materials, the laminating degree between the membrane layers is uneven in the assembling process, gaps exist, certain static electricity exists between the membrane materials, the sample infiltration process is longer, the reaction time is prolonged and the like are avoided.

Through setting up the siphon groove and realizing siphon application of sample, need not the membrane material and do reagent carrier or strain blood, convenient operation, it is little to need blood volume.

According to the preparation method of the blood fat three-item test strip, the reagent of the color development layer, the reaction layer, the blood filtering layer and the diffusion layer is sequentially arranged on the transparent substrate, and the corresponding layers are obtained through drying; the stability of the film forming process is good, and the uniformity of each layer of the test strip after film forming is high.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate certain embodiments of the present application and therefore should not be considered as limiting the scope of the present application.

FIG. 1 is a schematic diagram of a blood lipid three-item test strip according to an embodiment;

fig. 2 is a schematic diagram of a layered structure of a blood lipid three-item test strip according to an embodiment.

Reference numerals:

1-a transparent substrate; 2-a color layer; 3-a reaction layer; 4-a blood filtering layer; a 5-diffusion layer; 6-a siphon groove layer; 7-hydrophilic film layer.

Detailed Description

The term as used herein:

"prepared from … …" is synonymous with "comprising". The terms "comprising," "including," "having," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, step, method, article, or apparatus.

The conjunction "consisting of … …" excludes any unspecified element, step or component. If used in a claim, such phrase will cause the claim to be closed, such that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of … …" appears in a clause of the claim body, rather than immediately following the subject, it is limited to only the elements described in that clause; other elements are not excluded from the stated claims as a whole.

When an equivalent, concentration, or other value or parameter is expressed as a range, preferred range, or a range bounded by a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when ranges of "1 to 5" are disclosed, the described ranges should be construed to include ranges of "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a numerical range is described herein, unless otherwise indicated, the range is intended to include its endpoints and all integers and fractions within the range.

In these examples, the parts and percentages are by mass unless otherwise indicated.

"parts by mass" means a basic unit of measurement showing the mass ratio of a plurality of components, and 1 part may be any unit mass, for example, 1g may be expressed, 2.689g may be expressed, and the like. If we say that the mass part of the a component is a part and the mass part of the B component is B part, the ratio a of the mass of the a component to the mass of the B component is represented as: b. alternatively, the mass of the A component is aK, and the mass of the B component is bK (K is an arbitrary number and represents a multiple factor). It is not misunderstood that the sum of the parts by mass of all the components is not limited to 100 parts, unlike the parts by mass.

"and/or" is used to indicate that one or both of the illustrated cases may occur, e.g., a and/or B include (a and B) and (a or B).

Embodiments of the present application will be described in detail below with reference to specific examples, but it will be understood by those skilled in the art that the following examples are only for illustration of the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Examples

As shown in fig. 1 and 2, the present embodiment provides a blood lipid three-item test strip, which sequentially includes, from bottom to top: a transparent substrate 1, a color developing layer 2, a reaction layer 3, a blood filtering layer 4, a diffusion layer 5, a siphon groove layer 6 and a hydrophilic film layer 7; the color-developing layer 2 is divided into three parts, and is printed by cholesterol, high density lipoprotein and triglyceride reactant. Above the color-developing layer 2 are corresponding three different reaction layers 3 (or background-removed layers), above cholesterol and triglyceride are background layers, above HDL is a reaction layer containing precipitant (for convenience of expression, collectively referred to as reaction layer 3).

Wherein, the transparent substrate 1 is made of PVC hard board, and the transparency is more than or equal to 85%.

The chromogenic layer comprises an independent cholesterol chromogenic region, a high-density lipoprotein chromogenic region and a triglyceride chromogenic region; the cholesterol color development zone, the high density lipoprotein color development zone and the triglyceride color development zone are obtained by sequentially dripping a color development zone film forming agent, a color development agent and a color development zone isolating agent for solidification molding;

the reaction layer comprises an independent cholesterol reaction zone, a high-density lipoprotein reaction zone and a triglyceride reaction zone; the cholesterol reaction zone, the high-density lipoprotein reaction zone and the triglyceride reaction zone are all obtained by curing and molding a reaction zone film forming agent, a reaction reagent and a reaction zone isolating agent which are sequentially dripped from bottom to top;

the cholesterol color development zone is arranged corresponding to the cholesterol reaction zone, the high density lipoprotein color development zone is arranged corresponding to the high density lipoprotein reaction zone, and the triglyceride color development zone is arranged corresponding to the triglyceride reaction zone.

The preparation flow of the blood lipid three-item test strip is as follows:

1. scribing the transparent substrate 1 (pvc substrate) with hot melt adhesive to form three color layer tracks;

2. different color developing layer reagents (sequentially coating a color developing region film forming agent and a color developing agent) are printed on the corresponding color developing layer 2, and after airing, a color developing region isolating agent is coated and aired, the color developing layer 2 is obtained;

3. different reaction layer reagents (sequentially coating a reaction zone film forming agent and a reaction reagent) are printed on the upper surface of the color developing layer 2, a layer of reaction zone isolating agent is coated after airing, and a reaction layer 3 is obtained after airing;

4. coating a blood filtering layer reagent above the reaction layer 3, and airing to obtain a blood filtering layer 4;

5. coating a diffusion layer reagent on the upper part of the blood filtering layer 4, and airing to obtain a diffusion layer 5;

6. assembling a siphon groove to obtain a siphon groove layer 6, and then assembling a hydrophilic film to obtain a hydrophilic film layer 7;

7. cutting to obtain three blood lipid test strips.

The three blood fat test strips are simple to prepare, convenient to sample and small in sample adding amount; makes up for the shortages of dry chemical test strips.

The sample reaction principle and flow of the blood fat three-item test strip are as follows:

the blood sample enters the diffusion layer 5 through the siphon grooves of the siphon groove layer 6, uniformly diffuses to the blood filtering layer 4, blood cells are filtered and trapped, and the serum sample sequentially enters the reaction layer 3 and the color developing layer 2 to generate color development reaction.

The three different partitions of the color-developing layer 2 and the reaction layer 3 are not limited to the juxtaposed structure shown in fig. 2, and may be arranged in a "delta" shape or other suitable structure.

1. Synthesis of retroreflective material 20g of commercially available titanium dioxide particles were dispersed in 200 mL ethanol, 0.5 mL ammonia (28 wt%) was added and sonicated for 15 min. TBOT (butyl titanate) was then added thereto and the reaction was stirred for 24 hours. Then, the mixture was centrifuged, washed with water and ethanol 3 times each, and the obtained powder was dried in a box at 50℃for 48 hours. Finally, the resulting sample was treated at 500 ℃ for 2 hours to remove organic species and increase the crystallization degree.

Barium sulfate was treated in a similar manner to titanium dioxide.

2. Synthesis of mesoporous silica:

measuring 115 mL ethanol and 30 mL ammonia water, magnetically stirring, dropwise adding TEOS after 10 minutes, reacting at a certain temperature, and synthesizing SiO with different particle sizes and forms by adjusting the amount of TEOS and the stirring temperature and speed ₂ 。

Description of each layer of components:

color development film forming agent: cellulose acetate 40 g/m ³ First white reflective material (titanium dioxide) 200g/m ³ Self-made silica 200g/m as described above ³ Diatomite 400g/m ³ 。

The color development zone separating agent is gelatin 10g/m ³ ；

The developer components and contents of the cholesterol color development zone, the high density lipoprotein color development zone and the triglyceride color development zone are shown in Table 1 below:

TABLE 1 color developer composition and content

The reaction zone film former comprises 20g/m cellulose acetate ³ 100g/m of the self-made titanium dioxide white reflecting material ³ 、OP-10 5g/m ³ After the film formation layer was dried, the following reagents were printed in different reaction areas.

The isolation agent in the reaction area is gelatin, and the dosage is 20g/m ³ 。

The reactant composition and content of the different reaction zones are shown in table 2 below:

TABLE 2 reactant composition and content

Blood filtering layer 4:

the blood filtering layer comprises cellulose acetate 10g/m ³ Titanium dioxide 100g/m ³ Diatomite 100g/m ³ The artificially synthesized SiO ₂ Mesoporous material 100g/m ³ Canavalia gladiata lectin A5 g/m ³ ；

Diffusion layer 5 composition:

cellulose acetate 40 g/m ³ The self-made TiO ₂ 100 g/m ³ Diatomite 100g/m ³ Self-made SiO as described above ₂ Material 200g/m ³ Triton 10g/m ³ 。

Comparative example 1 structural comparison

Conventional test strip structures and methods (made according to patent application CN 202222285116.7) and sample application patterns were compared with test strips provided in the examples of the present application. 20 volunteers were collected, each volunteer was tested and compared with the blood lipid test strip provided in the examples of the present application and comparative example 1, the specific steps being:

s1, sterilizing by using an alcohol sheet, S2, pricking fingertips by using a blood taking needle, and S3: the first drop of blood sample is wiped off with a cotton swab, S4: blood sampling tests were performed according to the instructions.

And observing the simplicity and the whole blood sampling amount of the test process, and collecting opinion feedback after the volunteers use.

Results:

the traditional structure is as follows: after peripheral blood sampling, the sample is sucked by a capillary tube and transferred to a test strip, and the sample adding amount is 35-40 microliters.

The new structure of this application: and (3) adopting siphon sample adding, wherein the sample adding amount is less than 20 microlitres.

The traditional structure is as follows: the collected sample size is large, and the fingers of a subject need to be extruded; transfer after pipetting is required.

The new structure is as follows: the sample adding mode is simpler and more convenient: the sample adding amount is small, and the fingers do not need to be squeezed; only the siphon port of the test strip is required to be aligned with the bleeding point, and sample transfer is not required.

The details are shown in table 3 below:

table 3 experience results

Conclusion: by contrast, with the conventional structure, the sampling mode is more complex: when transferring a sample, the capillary is used for sucking the sample, the sample is easy to interrupt, and the sample adding quantity is controlled inaccurately; the sample adding amount is large; the peripheral blood volume is small, the bleeding volume is about 20 microliters, and when the traditional test strip (comparative example 1) needs to collect 35-40 microliters of samples, the requirement of blood consumption needs to be met in an extrusion mode, so that the user experience is poor.

Therefore, the traditional physical stacking of the film materials is replaced by the printing mode, the adding amount is obviously reduced, the siphon structure is matched, the sample adding is convenient, the value outlet time is shortened, and the experience of a subject is better.

Comparative example 2

Performance comparison of New test strips with existing test strips (made according to patent application CN 202222285116.7)

1. Taking the test strip and the commercial test strip for performance comparison experiments:

1.1 Accuracy comparison

The test strip provided in the example and a commercially available test strip are taken to test 20 clinical samples, each test is carried out for 1 time, and the test values are respectively compared with the test results of the biochemical analyzer.

1.2 Precision comparison

1.2.1 intra-batch differential contrast experiments

And testing the same quality control serum sample, continuously testing each batch of test strips for 20 times, counting test results, and analyzing the precision in the batch.

The total mean and standard deviation were calculated from the 20 data collected, and any difference between the result and the total mean was considered an outlier when it exceeded 4 standard deviations.

1.2.2 Batch-to-batch differential comparison experiment:

control 1 and example strips were selected 2 batches each, and quality control serum samples of the same concentration were tested, two sets of data per day, for 20 consecutive days.

In carrying out this evaluation work, it is necessary for the same or a group of operators to carry out on the same instrument, and the same calibrator, reagents of the same type and lot number should be used. The time is not less than 20 working days.

2. Analysis of results:

2.1 The accuracy results are shown in table 4 below:

table 4 accuracy comparison results

2.2 Precision in batch:

the batch precision of the example strip was slightly better than that of comparative example 1, and the results are shown in Table 5 below:

table 5 results of intra-lot difference comparisons for two structures

2.3 precision between batches

The batch to batch precision of the example strip was better than that of comparative example 2, and the results are shown in tables 6, 7 and 8 below:

TABLE 6 results of comparative example 2 Structure batch-to-batch differential test

Table 7 example batch-to-batch difference test results

Table 8 results of comparison of the differences between two test strip lots

Conclusion: the film forming reagents adopted by the test strip are all basic reagents, the film forming process is controlled strictly, and the color development layer of the blood fat test strip after film forming is uniform; the film materials used in the commercial test strips of comparative example 1 generally originate from different film material manufacturers, and the difference between the material batches is slightly larger due to the difference of the transportation and storage conditions, in addition, the lamination degree between the film layers cannot be controlled in the assembly and cutting processes, the electrostatic effect between the film layers is different due to the different humidity of the assembly environment, and the large difference between the batch of the commercial test strips may be caused due to the reasons.

Comparative example 3

The test cards provided by application numbers CN202222285116.7 and CN202010828814.X are used as controls.

The two patent structures are physical lists of film materials and are bonded by PET double-sided adhesive tape. Considering that certain gaps exist between the membrane layers due to electrostatic action or crimping force between the membrane materials, samples are trapped between the gaps, and the outside of the test strip is fixed by a plastic clamping shell, but the retention rate of the samples between the membrane materials is still higher, so that the consumption of the samples is higher, in addition, the membrane materials are generally expensive, and the production and assembly processes are complex.

According to the scheme provided by the application, different functional layers are directly formed into the film in a printing, spraying or coating mode, so that the pores caused by physical superposition are reduced, the structure is compact, and the required sample size is reduced; through the chemical action between the simple materials, the microstructure which is suitable for the needs of different functional areas is formed, the reagent is prepared according to the process, then printing, spraying or coating is carried out to form a layer of composite film, and a siphon structure is added on the composite film, so that three blood fat test strips are formed, and the problems of the traditional blood fat three-in-one product are solved.

Comparative example 4

Unlike the examples, commercially available titania and silica materials were used.

Comparison of hemofiltration performance:

taking the same venous blood sample, and using the blood lipid concentration of the biochemical analyzer test sample as the true value concentration; samples were prepared for different hematocrit samples as follows: 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% hct. The test was performed separately with the test strips of comparative example 4 and example, and the deviations of the test concentration values of the different hematocrit samples from the biochemical test results were compared. The results are shown in tables 9 and 10 below:

TABLE 9 anti-hematocrit test results

TABLE 10 comparison of anti-hematocrit Performance

Conclusion: compared with the comparative example 4, the surface of the reflecting material and the surface of the absorbing material in the film forming reagent used in the test strip are modified, so that the film layer has good biocompatibility and better diffusion performance, and the blood filtering functional area has better absorption on blood cells.

Comparative example 5

Unlike the examples, the color-development-region separating agent and the reaction-region separating agent were not added when the color-development layer and the reaction layer were prepared.

1. Accuracy assessment:

the test strips of example and comparative example 5 were used to test 20 clinical samples, each 1 time, and the test values were compared with the biochemical analyzer test results, respectively. The results are shown in Table 11:

TABLE 11 accuracy test results

2. Acceleration stability assessment:

test strips of examples and comparative example 4 were taken and subjected to accelerated aging at 55℃for 5d, 10d, 15d and 20d, respectively, and the same serum samples were tested 2 times each, and the test average values were compared with the biochemical analyzer test results, respectively. The results are shown in Table 12:

TABLE 12 accelerated stability results

The accuracy results can be seen: the test results of comparative example 5 (no isolation layer) were lower than the biochemical values, more than 20% deviation, and worse than those of the examples (isolation layer).

After accelerated aging treatment at 55 ℃, the test result of comparative example 5 is rapidly reduced, HDL item is failed after 15 days, and the accelerated stability of the test strip is poor.

Conclusion: the protective effect of the isolating agent is not generated, the film forming agent covers the reaction agent in the process of coating, the active substances in the reaction agent can be damaged, the test signal is reduced, the isolating agent is added, and the protective effect on the reaction agent in the film forming process is realized. In the acceleration treatment process of the test strip, the isolating agent also plays a role in protecting the reaction reagent, and the stability is good.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the present application has been described in detail with reference to the foregoing embodiments, it should be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present application.

Furthermore, those skilled in the art will appreciate that while some embodiments herein include some features but not others included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the present application and form different embodiments. For example, in the claims below, any of the claimed embodiments may be used in any combination. The information disclosed in this background section is only for enhancement of understanding of the general background of the application and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Claims (10)

1. The blood fat three-item test strip is characterized by comprising a transparent substrate, a color developing layer, a reaction layer, a blood filtering layer, a diffusion layer, a siphon groove layer and a hydrophilic film layer which are sequentially laminated from bottom to top;

the chromogenic layer comprises an independent cholesterol chromogenic region, a high-density lipoprotein chromogenic region and a triglyceride chromogenic region; the cholesterol color development zone, the high density lipoprotein color development zone and the triglyceride color development zone are formed by sequentially arranging a color development zone film forming agent, a color development agent and a color development zone isolating agent from bottom to top;

the reaction layer comprises an independent cholesterol reaction zone, a high-density lipoprotein reaction zone and a triglyceride reaction zone; the cholesterol reaction zone, the high-density lipoprotein reaction zone and the triglyceride reaction zone are formed by sequentially arranging a reaction zone film forming agent, a reaction reagent and a reaction zone isolating agent from bottom to top;

the cholesterol color development zone is arranged corresponding to the cholesterol reaction zone, the high-density lipoprotein color development zone is arranged corresponding to the high-density lipoprotein reaction zone, and the triglyceride color development zone is arranged corresponding to the triglyceride reaction zone;

the color developing layer, the reaction layer, the blood filtering layer and the diffusion layer have porous structures and are formed by the reagents in the corresponding layers.

2. The blood lipid three test strip of claim 1, wherein the chromogenic zone film former comprises 20-60g/m cellulose acetate ³ 40-400g/m of first white reflecting material ³ 40-400g/m diatomite ³ Microporous material 40-400g/m, natural and/or synthetic ³ ；

The first white reflecting material is titanium dioxide and/or barium sulfate.

3. The blood lipid three test strip of claim 1, wherein the color zone separating agent comprises gelatin, xanthan gum, agar and derivatives thereof in an amount of 5-50 g/m ³ ；

The reaction zone separating agent comprises gelatin, xanthan gum, agar and its derivatives, and the content is 5-50 g/m ³ 。

4. The blood lipid three test strip of claim 1, wherein the cholesterol color development zone, the high density lipoprotein color development zone, and the color development reagent of the triglyceride color development zone each independently comprise a first color development reagent 0.5-5mM, a first buffer 50-1000mM, a first surfactant 0.1-2g/L, a first protective agent 0.5-5mM, and a first preservative 0.1-5.0 g/L;

the first color reagent comprises one or more of N- (2-hydroxy-3-sulfopropyl) -3', 5-dimethoxy aniline sodium salt, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt and 4-aminoantipyrine;

the buffer solution comprises any one of phosphate buffer solution, tris-HCl buffer solution or 3- (N-morpholinyl) propane sulfonic acid buffer solution, and the pH value is 6.5-7.5;

the first surfactant comprises one or more of laureth-35, polyethylene glycol octadecyl ether, sodium dodecyl benzene sulfonate, tween-20, tween-40, tween-60, triton-100, triton-405 and OP-10;

the first protective agent comprises one or more of bovine serum albumin, trehalose, fish gelatin, mannitol and polyvinyl alcohol;

the first preservative comprises one or more of sodium azide, sodium benzoate, proclin 300, krovin-100.

5. The blood lipid three test strip of claim 1, wherein the reaction zone film former comprises cellulose acetate 10-50g/m ³ Second white reflecting material 20-200g/m ³ Second surfactant 2-10 g/m ³ ；

The second white reflecting material is titanium dioxide and/or barium sulfate;

the second surfactant comprises one or more of laureth-35, polyethylene glycol octadecyl ether, sodium dodecyl benzene sulfonate, tween-20, tween-40, tween-60, triton-100, triton-405 and OP-10.

6. The three blood lipid test strip of claim 1, wherein the reagents of the cholesterol reaction zone comprise cholesterol oxidase 10-200KU/L, cholesterol esterase 10-100KU/L, second buffer solution 50-500mM, second protectant 0.5-10mM, third surfactant 0.1-5.0g/L and second preservative 0.5-5.0g/L, the reagents of the high density lipoprotein reaction zone comprise cholesterol oxidase 10-100KU/L, cholesterol esterase 10-50KU/L, third buffer solution 50-500mM, third protectant 0.5-10mM, precipitant 1-5g/L, fourth surfactant 0.1-5.0g/L and third preservative 0.5-5.0g/L, and the reagents of the triglyceride reaction zone comprise glycerol kinase 50-200 KU/L, fourth buffer solution 50-500mM, fourth protectant 0.5-5 g/L, fifth surfactant 0.1-5.0g/L and fourth preservative 0.5-5 mM;

the second buffer solution, the third buffer solution and the fourth buffer solution respectively and independently comprise any one of a phosphate buffer solution, a Tris-HCl buffer solution or a 3- (N-morpholinyl) propane sulfonic acid buffer solution, the pH value of the second buffer solution is 6.5-7.0, the pH value of the third buffer solution is 6.5-7.0, and the pH value of the fourth buffer solution is 7.0-7.5;

the second protective agent and the third protective agent respectively independently comprise one or more of bovine serum albumin, trehalose and glycerol, and the fourth protective agent comprises one or more of serum albumin, trehalose, polyvinylpyrrolidone and polyvinyl alcohol;

the third surfactant, the fourth surfactant and the fifth surfactant independently comprise one or more of laureth-35, polyethylene glycol octadecyl ether, sodium dodecyl benzene sulfonate, tween-20, tween-40, triton-405 and OP-10;

the precipitant comprises one or more of phosphotungstic acid, dextran sulfate and polyethylene glycol;

the second preservative, the third preservative and the fourth preservative each independently comprise one or more of sodium azide, sodium benzoate, proclin 300 and Krovin-100.

7. The blood lipid three test strip of claim 1, wherein the blood filtering layer comprises cellulose acetate 2-20g/m ³ 40-400g/m of third white reflecting material ³ 40-400g/m diatomite ³ Natural and/or synthetic microporous materials 20-200g/m ³ Lectin 1-10g/m ³ ；

The third white reflecting material is titanium dioxide and/or barium sulfate, and the lectin comprises one or more of Canavalia gladiata lectin A, wheat germ element and soybean lectin; natural microporous materials include microporous cellulose and synthetic microporous materials include resins and/or silica.

8. The blood lipid three test strip of claim 1, wherein the diffusion layer comprises 20-60g/m cellulose acetate ³ Fourth white reflecting material 20-200g/m ³ Diatomite 100-500g/m ³ Microporous material of 100-500g/m, natural and/or artificial synthesis ³ Sixth surfactant 2-20g/m ³ ；

The fourth white reflecting material is titanium dioxide and/or barium sulfate, the natural microporous material comprises microporous cellulose, and the artificially synthesized microporous material comprises resin and/or silicon dioxide; the sixth surfactant comprises one or more of laureth-35, polyethylene glycol octadecyl ether, sodium dodecyl benzene sulfonate, tween-20, tween-40, triton-405 and OP-10.

9. The blood lipid three test strip of any one of claims 1-8, wherein the material of the transparent substrate comprises any one of polycarbonate, PET, PVC, PE.

10. A method of preparing the blood lipid three test strip of any one of claims 1-9, comprising:

sequentially arranging reagents of the color development layer, the reaction layer, the blood filtering layer and the diffusion layer on the transparent substrate, and drying and forming to obtain corresponding layers;

and setting the siphon groove layer and the hydrophilic film layer, and performing post-treatment to obtain the blood fat three-item test strip.

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