阅读说明：本技术 用于测定高密度脂蛋白胆固醇的方法 (Method for measuring high density lipoprotein cholesterol ) 是由 叶学松 梁波 自强 于 2019-09-17 设计创作，主要内容包括：用于测定高密度脂蛋白胆固醇的方法,包括以下操作：获得10微升体液样本,将体液样本加入上述试纸的加样区,等待反应,用荧光免疫分析仪测量试纸上的量子点线条的荧光强度,比对荧光强度-胆固醇浓度曲线获得高密度脂蛋白胆固醇含量。本发明利用双氧水淬灭量子点的方式来检测高密度脂蛋白的含量,精度高,所需样本量少的优点。(method for determining high density lipoprotein cholesterol comprising the following operations: and (3) obtaining a 10 microliter body fluid sample, adding the body fluid sample into the sample adding area of the test paper, waiting for reaction, measuring the fluorescence intensity of a quantum dot line on the test paper by using a fluorescence immunoassay analyzer, and comparing the fluorescence intensity with a cholesterol concentration curve to obtain the content of the high-density lipoprotein cholesterol. The method provided by the invention has the advantages of high precision and less required sample amount by detecting the content of the high-density lipoprotein in a hydrogen peroxide quenching quantum dot mode.)

1. method for determining high density lipoprotein cholesterol comprising the following operations: and obtaining a body fluid sample of 10 microliter or less than 10 microliter, adding the body fluid sample into the sample adding area of the test paper, waiting for reaction, measuring the fluorescence intensity of a quantum dot line on the test paper by using a fluorescence immunoassay analyzer, and comparing the fluorescence intensity with a cholesterol concentration curve to obtain the content of the high-density lipoprotein cholesterol.

2. The method for measuring high-density lipoprotein cholesterol according to claim 1, characterized in that: the test paper of the same batch is produced and obtained by the reagent configured at the same time, a plurality of test paper is randomly extracted, a high-density lipoprotein cholesterol standard substance with known concentration is tested, the fluorescence intensity on each test paper is measured by a fluorescence immunoassay analyzer, a map is drawn by taking the cholesterol concentration and the fluorescence intensity as coordinates respectively, the corresponding points of the cholesterol concentration-fluorescence intensity are corresponded to the map, all the corresponded points are fitted into a standard curve, and a relational formula corresponding to the standard curve is calculated; in actual use, the cholesterol concentration is obtained by the fluorescence intensity through a standard curve or a relational formula.

3. The method for measuring high-density lipoprotein cholesterol according to claim 1, characterized in that: the test paper used in the method comprises a separation layer, a reaction layer and a detection layer; the separation layer receives a sample and separates high-density lipoprotein from the sample, the high-density lipoprotein obtained by separation permeates into the reaction layer, and the product of the reaction layer comprises hydrogen peroxide; the hydrogen peroxide permeates into the detection layer, the detection layer comprises a diffusion part and quantum dot lines, and the hydrogen peroxide firstly permeates into the diffusion part and then diffuses into the quantum dot lines from the diffusion part.

4. The method for measuring high-density lipoprotein cholesterol according to claim 3, characterized in that: the test paper is provided with a plurality of quantum dot lines, and the fluorescence intensity average value of all the quantum dot lines is used as the fluorescence intensity of the current test paper.

Technical Field

The invention relates to a method for measuring high-density lipoprotein cholesterol, in particular to a test strip for measuring the high-density lipoprotein cholesterol and a preparation method thereof.

Background

POCT, point-of-care testing, refers to clinical testing and bedside testing performed near a patient, and is not always performed by a clinical laboratory technician. The method is a new method for analyzing the sample immediately on the sampling site, saving complex processing procedures of the sample during laboratory test and quickly obtaining test results. Along with the improvement of living standard of people, the health consciousness of people is strengthening, and POCT diagnosis equipment products are more and more favored by people. Because the POCT diagnosis product has small volume and simple operation, the accurate result can be obtained without strong professional of operators, which makes the detection of people at home possible. ,

The cholesterol can be matched with deputy protein in serum to form lipoprotein, and the lipoprotein can be classified into chylomicron, very low density lipoprotein, low density lipoprotein and high density lipoprotein. Epidemiologically, the level of HDL cholesterol is inversely correlated with the frequency of occurrence of arteriosclerotic diseases, and its detection is widely used for the prevention or diagnosis of ischemic heart diseases. The traditional methods for detecting HDL cholesterol are mainly to separate HDL by centrifugation or electrophoresis, and then perform subsequent operations, and these methods are often complicated and require a person with professional skills to complete the relevant operations, so these methods are not suitable for home use.

There have been related patents reporting products capable of rapidly detecting high density lipoprotein cholesterol, for example, patent (CN 1258601C) discloses a test piece for measuring High Density Lipoprotein (HDL) cholesterol, which uses two surfactants to achieve separation of High Density Lipoprotein (HDL) from non-high density lipoprotein, and the test piece has a simple structure and is convenient and rapid to detect. The patent (US 7374719B 2) realizes the simultaneous detection of total cholesterol, high density lipoprotein cholesterol and triglyceride by fixing functional layers processed by different reagents in a designed test paper card shell, and indirectly gives the amount of non-high density lipoprotein cholesterol by calculation, the method is fast and convenient, and the sample amount of 35 mu L is required.

Although the above reported products are all suitable for home rapid diagnosis, since the above products are all based on the color development principle, for example, the patent (US 7374719B 2) detects the color change of the generated product by light reflection, and then knows the amount of the detected object in the sample. Because the color development itself needs more products generated by the reaction layer to enable the color to reach a certain depth change for detection, the products need higher sample volume when being detected, and meanwhile, the color development itself relates to enzyme reaction, which increases the types and the dosage of the enzyme related to the products. Furthermore, the enzyme reaction is greatly affected by the sample matrix, so the product faces a larger "matrix effect", thereby causing a larger uncertainty of the detection result.

disclosure of Invention

in view of the above situation, in order to overcome the defects of the prior art, the present invention aims to provide a detection apparatus for detecting high-density lipoproteins by using a hydrogen peroxide quenching quantum dot manner. The detection device can separate high-density lipoprotein in a body fluid sample, so that the separated high-density lipoprotein is subjected to oxidation reaction to generate hydrogen peroxide, the hydrogen peroxide is diffused to quantum dot lines to generate carbon quantum dot fluorescence quenching reaction, and the carbon quantum dot fluorescence quenching reaction represents the content of the high-density lipoprotein, thereby realizing the purpose of testing the content of the high-density lipoprotein.

In a first aspect of the present invention, there is provided a high density lipoprotein cholesterol assay strip, comprising a separation layer, a reaction layer and a detection layer; the separation layer receives a sample and separates high-density lipoprotein from the sample, the high-density lipoprotein obtained by separation permeates into the reaction layer, and the product of the reaction layer comprises hydrogen peroxide; the hydrogen peroxide permeates into the detection layer, the detection layer comprises a diffusion part and quantum dot lines, and the hydrogen peroxide firstly permeates into the diffusion part and then diffuses into the quantum dot lines from the diffusion part.

Preferably, the separation layer is on top of the reaction layer, which is on top of the detection layer. The sample permeates downwards layer by layer under the action of gravity.

Preferably, in a bottom view direction, the reaction layer completely covers the separation layer; in a plan view direction, the reaction layer partially covers the diffusion portion of the detection layer, and the quantum dot lines are located at portions not covered by the reaction layer. The hydrogen peroxide generated by the reaction layer firstly permeates into the diffusion part and then diffuses from the diffusion part to the quantum dot line. The quantum dot lines are arranged outside the reaction layer and the separation layer, so that the quantum dot lines can be directly observed and tested. Of course, the quantum dot lines may be covered under the reaction layer and the separation layer as long as the fluorescence immunoassay analyzer can detect the quantum dot lines.

In some preferred embodiments, the test strip includes a filter layer from which the sample is introduced and through which the filtered sample permeates to the separation layer. The purpose of the filter layer is to filter blood cells, making the test strip suitable for use with whole blood samples. The test paper is not provided with a filter layer, and is suitable for body fluid samples except whole blood, such as blood plasma and the like.

In some preferred embodiments, the detection layer is a nitrocellulose membrane (Millipore 135) on which lines of quantum dots are scribed. Preferably, the quantum dot lines are at least 1. Preferably, the number of the quantum dot lines is 3.

preferably, the filter layer treatment agent formulation: sucrose: 50.0g/L, surfactant S9: 2.0g/L, Tween-20: 1.0g/L, polyvinylpyrrolidone K30: 5.0g/L, and 0.10g/L of dipotassium ethylenediaminetetraacetate, were prepared using 0.01mol/L PBS (pH 7.4).

In some preferred forms, the blood filtration layer treatment reagent formulation comprises: sucrose: the concentration of the active carbon is 50.0g/L,

Surfactant S9: 2.0 g/L;

Tween-20: 1.0 g/L;

Polyvinylpyrrolidone K30: 5.0 g/L;

ethylene diamine tetraacetic acid dipotassium salt: 0.1 g/L;

Prepared using 0.01mol/L PBS (pH7.4)

In some preferred embodiments, the high density lipoprotein separation layer treatment reagent formulation:

magnesium sulfate: 5.0 g/L;

Phosphotungstic acid: 50.0 g/L;

Sorbitol: 10.0 g/L;

Sucrose: the concentration of the active carbon is 50.0g/L,

prepared using 0.01mol/L PBS (pH 6.5)

in some preferred embodiments, the formulation of the high density lipoprotein reaction layer treatment reagent:

triton X-100: 3.0 g/L;

Bovine serum albumin: 1.0 g/L;

Gantrez AN139：10.0g/L；

Sucrose: 50.0 g/L;

Cholesterol oxidase: 70.00 KU/L;

Cholesterol esterase: 180.00 KU/L;

polyvinylpyrrolidone-K30: 5.0 g/L;

Disodium ethylene diamine tetraacetate: 10.0 g/L;

Proclin300 1.0g/L；

this was formulated using 0.01mol/L PBS (pH 5.5).

in some preferred embodiments, a water-impermeable substrate is disposed below the detection layer, and the detection layer is fixed to the substrate. Preferably, the reaction layer, the separation layer and the filter layer are arranged on the detection layer by layer, and the layers are pressed and fixed by the cover. The pressing and fixing means that all the layers are mutually attached under the action of pressure, and in the process of use, storage or transportation and the like, all the layers do not displace mutually. The connection of face lid and base plate can adopt the connected mode of the face lid and the base plate of current test paper product, only need cover on the face and set up the position that compresses tightly reaction layer, separation layer and filter layer can, for example compress tightly the four corners of each layer with the post, or compress tightly the edge of each layer with the sand grip. Preferably, the face cover is provided with a sample adding hole and a display window, the quantum dot lines are positioned on the display window, and the part of the filter layer for receiving the sample is exposed out of the sample adding hole. That is, the test paper may be completely exposed to the sample hole, or only partially exposed to the sample hole. When the quantum dot lines are covered by the reaction layer, holes for exposing the quantum dot lines are arranged below the substrate, or after test paper detection is completed, the surface cover is disassembled, the detection layer (or the detection layer and the substrate) is taken out, the quantum dot lines are exposed, and fluorescence detection is carried out.

the invention combines the chromatography and quantum dot quenching principle, and has a blood filtering layer, a high-density lipoprotein separating layer, a high-density lipoprotein reaction layer and a detection layer marked with quantum dot lines. The test paper strip uses a PVC rubber plate as a substrate as a support body, wherein the detection layer marked with quantum dot lines is pasted on the PVC rubber plate support body, other layers are naturally accumulated on the detection layer marked with the quantum dot lines according to a set sequence, and a fixed distance is arranged at intervals of the detection layer on the detection layer. The layers are in intimate contact with each other by pressure provided by the face cover, wherein the cartridge may be directly used with a fluorescence immunochromatographic cartridge. Due to the adoption of the fluorescence quenching principle, the test strip only needs a small amount of samples and can be used for whole blood, serum and body fluid samples possibly containing high-density lipoprotein.

In a second aspect, the present invention is directed to a method of making the above test strip. The method comprises the following steps: manufacturing a detection layer: preparing a detection layer reagent, adding the detection layer reagent onto the detection layer, wetting the detection layer, and drying the detection layer with the detection layer reagent; drawing quantum dot lines on the dried detection layer; preparing a separation layer: preparing a separation layer reagent, adding the separation layer reagent to the separation layer, wetting the separation layer, and drying the separation layer with the separation layer reagent; preparing a reaction layer, adding a reaction layer reagent onto the reaction layer to wet the reaction layer, and drying the reaction layer with the reaction layer reagent; the detection layer, the reaction layer and the separation layer can be manufactured simultaneously or sequentially; placing the reaction layer on the detection layer, wherein the quantum dot lines are positioned outside the area covered by the reaction layer; the separation layer is overlapped with the reaction layer and is positioned above the reaction layer; and the separation layer, the reaction layer and the detection layer are compressed, and the sample adding part and the quantum dot lines are exposed.

preferably, the filter layer is manufactured, a filter layer reagent is added on the filter layer to wet the filter layer, and the filter layer with the filter layer reagent is dried; the filter layer is laminated on the separation layer, and the filter layer, the separation layer, the reaction layer and the detection layer are compressed.

Preferably, the detection layer is a nitrocellulose membrane. Preferably, the separation layer is a core silicon valley qualitative filter paper. Preferably, the reaction layer is a glass fiber membrane.

in some preferred forms, the cover and the substrate are formed, and the detection layer, the reaction layer and the separation layer, and/or the filter layer are compressed between the cover and the substrate.

in a third aspect, the present invention is directed to a method for measuring high density lipoprotein cholesterol using the test strip.

Method for determining high density lipoprotein cholesterol comprising the following operations: and (3) obtaining a 10 microliter body fluid sample, adding the body fluid sample into the sample adding area of the test paper, waiting for reaction, measuring the fluorescence intensity of a quantum dot line on the test paper by using a fluorescence immunoassay analyzer, and comparing the fluorescence intensity with a cholesterol concentration curve to obtain the content of the high-density lipoprotein cholesterol.

preferably, the test paper of the same batch is produced by the reagent configured at the same time, a plurality of test paper are randomly extracted, the high-density lipoprotein cholesterol standard substance with known concentration is tested, the fluorescence intensity on each test paper is measured by a fluorescence immunoassay analyzer, the cholesterol concentration and the fluorescence intensity are respectively taken as coordinates to draw a map, the cholesterol concentration-fluorescence intensity corresponding points correspond to the map, all the corresponding points are fitted into a standard curve, and a relational formula corresponding to the standard curve is calculated; in actual use, the cholesterol concentration is obtained by the fluorescence intensity through a standard curve or a relational formula.

Preferably, the test paper is provided with a plurality of quantum dot lines, and the fluorescence intensity mean value of all the quantum dot lines is used as the fluorescence intensity of the current test paper.

the invention changes the detection principle of color development, and uses a quantum dot which can be quenched by hydrogen peroxide and is an oxidation product of high-density lipoprotein cholesterol to detect the amount of the high-density lipoprotein cholesterol in a detected object. Because the quantum dots can be quenched by trace hydrogen peroxide, sensitive detection on target substances can be realized by using a small sample amount. Meanwhile, because the enzyme for developing color is removed, the possibility that the test paper is influenced by the sample substrate is reduced.

compared with the prior art, the invention has the following advantages:

(1) The test paper uses a small sample volume of 10 mu L, is convenient and quick to operate, can realize quick and accurate detection on the high-density lipoprotein cholesterol without the need of operators having high professional levels, and simultaneously reduces the influence on the detection result due to the difference of sample matrixes because of avoiding using chromogenic enzyme.

(2) the test paper can be assembled by using the existing fluorescence immunochromatographic card shell without flexible design.

(3) The test paper can finish rapid detection by using the existing fluorescence immunoassay analyzer

(4) After the reaction zone of the test strip is treated by different functional enzymes, the test strip can be used for detecting all substances which can generate hydrogen peroxide after being treated by the enzymes in principle, such as triglyceride, total cholesterol, creatinine and the like.

Description of the drawings:

FIG. 1: example 1 a test strip standard curve was prepared.

FIG. 2: example 2 a test strip standard curve was prepared.

FIG. 3: example 3 a test strip standard curve was prepared.

FIG. 4: example 4 a test strip standard curve was prepared.

FIG. 5: example 4 comparison of the test strip prepared in example 4 with the results of the Beckmann biochemical analyzer.

FIG. 6: test paper strip structure chart.

the labels in the figure are: 1: a filter layer; 2: a separation layer; 3: a reaction layer; 4: a detection layer; 5: a substrate; 6: quantum dot line

Detailed Description

the invention is explained in detail with reference to the drawings:

as shown in fig. 6, the high density lipoprotein cholesterol assay test paper includes a separation layer 2, a reaction layer 3 and a detection layer 4; the separation layer 2 receives a sample and separates high-density lipoprotein from the sample, the high-density lipoprotein obtained by separation permeates into the reaction layer 3, and the product of the reaction layer 3 comprises hydrogen peroxide; the hydrogen peroxide permeates into the detection layer 4, the detection layer 4 comprises a diffusion part and quantum dot lines 6, and the hydrogen peroxide firstly permeates into the diffusion part and then diffuses into the quantum dot lines 6 from the diffusion part.

The separation layer 2 is on the reaction layer 3, and the reaction layer 3 is on the detection layer 4. The sample permeates downwards layer by layer under the action of gravity.

In some embodiments, in a bottom view direction, the reaction layer 3 completely covers the separation layer 2; in the plan view, the reaction layer 3 partially covers the diffusion portion of the detection layer 4, and the quantum dot lines 6 are located at the portions not covered by the reaction layer 3. The hydrogen peroxide generated in the reaction layer 3 firstly permeates into the diffusion part and then diffuses from the diffusion part to the quantum dot line. The quantum dot lines 6 are arranged outside the reaction layer 3 and the separation layer 2, so that the quantum dot lines 6 can be observed and checked directly. Of course, the quantum dot lines 6 may be covered under the reaction layer 3 and the separation layer 2 as long as the fluorescence immunoassay analyzer can detect the quantum dot lines 6.

in some preferred modes, the test paper comprises a filter layer 1, a sample is added from the filter layer 1, and the filtered sample permeates into a separation layer 2. The purpose of filter layer 1 is to filter blood cells so that the test strip can be applied to a whole blood sample. The test paper does not need to be provided with the filter layer 1, and is suitable for body fluid samples other than whole blood, such as plasma and the like.

in some preferred embodiments, the detection layer 4 is a nitrocellulose membrane (Millipore 135) on which the lines 6 of quantum dots are scribed. Preferably, the quantum dot lines 6 are at least 1. Preferably, the number of the quantum dot lines 6 is 3.

In some preferred embodiments, a water-impermeable substrate 5 is disposed below the detection layer 4, and the detection layer 4 is fixed on the substrate 5. Preferably, the reaction layer 3, the separation layer 2 and the filtration layer 1 are placed on the detection layer 4 layer by layer, and the layers are pressed and fixed by the cover. The pressing and fixing means that all the layers are mutually attached under the action of pressure, and in the process of use, storage or transportation and the like, all the layers do not displace mutually. The connection of the surface cover and the substrate 5 can adopt the connection mode of the surface cover and the substrate 5 of the existing test paper product, and only the parts for compressing the reaction layer 3, the separation layer 2 and the filter layer 1 are required to be arranged on the surface cover, for example, four corners of each layer are compressed by columns, or edges of each layer are compressed by raised lines. Preferably, the face cover is provided with a sample adding hole and a display window, the quantum dot line 6 is positioned on the display window, and the part of the filter layer 1 for receiving the sample is exposed out of the sample adding hole. That is, the test paper may be completely exposed to the sample hole, or only partially exposed to the sample hole. When the quantum dot lines 6 are covered by the reaction layer 3, holes for exposing the quantum dot lines 6 are arranged below the substrate 5, or after the test paper is detected, the surface cover is disassembled, the detection layer 4 is taken out (or the detection layer 4 and the substrate 5 are together), the quantum dot lines 6 are exposed, and fluorescence detection is carried out.