A homocysteine detection device

Abstract

The application discloses a homocysteine detection device, and belongs to the field of in vitro diagnostic reagents, which comprises an upper layer, a diffusion layer, a blood filtration membrane, a reaction membrane and a bottom layer. The upper layer is provided with a sample adding hole for sample adding. The bottom layer is provided with a detection hole for detection. The reaction membrane contains L-serine, cystathionine beta-synthase, cystathionine beta-lyase, pyruvate oxidase, pyridoxal 5'-phosphate, peroxidase and a color developing agent. The reaction membrane further contains 3', 4, 4', 5, 5', 6-hexahydroxy-3, 6'-oxybis[tetrahydro-2H-pyran-2-methanol] octoacetate. The homocysteine detection device provided by the application can inhibit non-specific background color development and improve detection accuracy.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics, and in particular relates to a device for detecting homocysteine. Background Technology

[0002] Homocysteine ​​(Hcy), also known as homocysteine, is a sulfur-containing amino acid and an intermediate formed in the metabolic bypass pathway of cystathionine and methionine transsulfurization and methylation. Similar to cysteine, it is easily spontaneously oxidized, and two homocysteine ​​molecules form homocysteine ​​with disulfide bonds.

[0003] Homocysteine ​​exists in the human body mainly in the form of reduced form, cystine (oxidized form), homocysteine-homocysteine ​​and homocysteine-cystine disulfide mixed oxidized form. In plasma, there are two forms: free and protein-bound. The former accounts for 20%, while the latter is bound to albumin and accounts for 70% to 80%.

[0004] High levels of homocysteine ​​are closely associated with a variety of diseases. Defects in the synthesis and metabolism of homocysteine, as well as related enzyme systems and nutritional deficiencies (folic acid, vitamin B2, and vitamin B6), can all cause hyperhomocysteinemia (which is easily corrected with treatment using folic acid, vitamin B6, vitamin B12, betaine, etc.). In addition, homocysteine ​​can cause damage to vascular endothelial cells, promote the proliferation of vascular smooth muscle cells, stimulate low-density lipoprotein oxidation, enhance platelet coagulation function, and promote thrombus formation, thereby leading to cardiovascular diseases and stroke, venous thrombosis, recurrent miscarriage, birth defects, neural tube defects, Alzheimer's disease, and other conditions.

[0005] Methods for detecting homocysteine ​​include high-performance liquid chromatography (HPLC), mass spectrometry (MS), and capillary electrophoresis. However, these methods involve expensive equipment, require specialized personnel, and are time-consuming. Clinically, cyclic enzymatic methods are commonly used for homocysteine ​​detection, and the cystathionine route cyclic enzymatic method is widely used clinically due to its limited number of coupled enzymes. The detection cycle is as follows: After the above two steps of reaction, homocysteine ​​remains in the cycle and is not consumed, while pyruvate is produced in large quantities. The overall reaction is as follows: The rate of pyruvate production is directly proportional to the concentration of homocysteine. By detecting the produced pyruvate, low concentrations of homocysteine ​​can be detected.

[0006] The following methods can be used to detect pyruvate. By reading the color changes with an instrument and applying a certain algorithm, the concentration of homocysteine ​​in the sample can be obtained.

[0007] However, during the above detection process, the cystathionine β-lyase involved in the reaction will undergo a non-specific reaction with L-serine to generate pyruvate, which leads to an increase in non-specific background value and causes inaccurate detection. Summary of the Invention

[0008] This invention provides a homocysteine ​​detection device with low background.

[0009] This invention provides a homocysteine ​​detection device, comprising an upper layer, a diffusion layer, a blood filtration membrane, a reaction membrane, and a bottom layer. The upper layer has sample application wells for sample application, and the bottom layer has detection wells for detection. The reaction membrane contains L-serine, cystathionine β-synthetase, cystathionine β-lyase, pyruvate oxidase, thiamine pyrophosphate, peroxidase, and a chromogenic reagent.

[0010] Preferably, the blood filtration membrane contains tris(2-carboxyethyl)phosphine (TCEP).

[0011] Preferably, the colorimetric agent is sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS) and 4-aminoantipyrine.

[0012] Preferably, the reaction membrane contains pyridoxal phosphate (PLP) and flavin adenine dinucleotide (FAD).

[0013] Preferably, the reaction membrane contains 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetic acid ester.

[0014] Preferably, the mass concentration of the 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetic acid ester is 0.001. 0.005%.

[0015] The 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetate inhibits the non-specific reaction between cystathionine β-lyase and serine, reducing the reaction background.

[0016] Preferably, the reaction membrane further contains a pyrido[2,3-b]pyrazine derivative.

[0017] Preferably, the pyrido[2,3-b]pyrazine derivative is 3-chloropyrido[2,3-b]pyrazine, 7-bromo-8-methylpyrido[2,3-b]pyrazine, or 2,3-dichloropyrido[2,3-b]pyrazine.

[0018] Preferably, the mass concentration of the pyrido[2,3-b]pyrazine derivative is 0.01%. 0.05%.

[0019] The addition of the pyrido[2,3-b]pyrazine derivative further inhibited the non-specific reaction between cystathionine β-lyase and serine, further reducing the reaction background.

[0020] Preferably, the blood filtration membrane is Topfan 1660, and the reaction membrane is Topfan nylon membrane Biodyne C 0.45μm.

[0021] Whole blood samples are added to the sample wells and, under the action of the diffusion layer, are evenly distributed onto the filtration membrane. Blood cells in the sample are retained by the filtration membrane, while tris(2-carboxyethyl)phosphine in the membrane is re-dissolved by the sample, and bound homocysteine ​​is dissociated into a free state. Next, the plasma permeates downwards to wet the reaction membrane, re-dissolving various reagents within it. The free homocysteine ​​reacts with L-serine under the action of cystathionine β-synthase and cystathionine β-lyase to generate pyruvate. Pyruvate then induces color development in the chromogenic reagent under the action of pyruvate oxidase and peroxidase. After a certain time, the color depth (i.e., reflectance) is read, and the homocysteine ​​concentration is obtained using an algorithm.

[0022] The detection device of this invention can be used with small detection instruments, such as a spectrophotometer, preferably a reflectance spectrophotometer. This spectrophotometer can detect the change in reflected light density at a specific wavelength after the homocysteine ​​detection device reacts, and the homocysteine ​​concentration is given by an algorithm. Because the homocysteine ​​detection device of this invention can eliminate the influence of non-specific color development on the detection, the detection can be more accurate. Furthermore, the detection device of this method is easy to operate, requiring no professional personnel, and can meet market demands under special conditions, especially for emergency and home applications. Compared with traditional methods using liquid biochemical reagents, the detection device of this invention can provide convenient and rapid testing for emergency departments, primary hospitals, homes, and small clinics. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the detection device of the present invention; Figure 2 This is a schematic diagram showing the drilling locations on the upper and lower layers of the present invention; Figure 3 This is a schematic diagram showing the location of the diffusion layer in this invention.

[0024] In the figure, the detection device is 10, the upper layer is 101, the bottom layer is 102, the diffusion layer is 103, the blood filtration membrane is 104, the reaction membrane is 105, the sample application port is 106, and the detection port is 107. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0026] It should be noted that when a component is said to be "located" on another component, it can be directly on the other component or there may be an intervening component. When a component is said to be "connected" to another component, it can be directly connected to the other component or there may be an intervening component.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] Example like Figure 1 As shown, the homocysteine ​​detection device 10 provided by the present invention includes an upper layer 101, a bottom layer 102, a diffusion layer 103, a blood filtration membrane 104, and a reaction membrane 105. The upper layer is provided with a sample application port 106, and the bottom layer is provided with a detection port 107.

[0029] The upper and lower layers can be made by attaching double-sided tape to a plastic substrate and then punching holes. Then, the treated diffusion layer, blood filtration membrane, and reaction membrane are placed in the middle, and the upper and lower layers are attached to form the homocysteine ​​detection device provided by this invention.

[0030] Example 1 Creating the top (bottom) layer: Take a 20cm x 8cm piece of double-sided tape with release paper on both sides, peel off one side of the release paper, and then neatly attach it to a 20cm x 8cm 0.35mm thick white PET sheet. Use a punching machine to punch 20 round holes with a diameter of 0.5mm into this PET sheet. The center of all the holes should be 20mm from the top of the sheet, and the distance between the centers should be 10mm. The center of the leftmost hole should be 5mm from the left edge of the sheet. Figure 2 As shown.

[0031] In this embodiment, the upper and lower layers are made of the same material. In actual products, the upper layer can be printed with arrows, project names, company names, etc.; the lower layer can be printed with black to reduce non-specific reflections. These are techniques well known to those skilled in the art.

[0032] Fabrication of the diffusion layer: A 20cm × 0.8cm PES 18 / 13 yarn from the Yarn Company was soaked in a 0.05% (v / v) Triton X100 solution for 10 minutes and then dried at 37°C to obtain the diffusion layer.

[0033] Preparation of blood filtration membrane: Cut the Sitopan membrane material 1660 into 20cm × 1cm pieces, and then add the following solution: Sodium dihydrogen phosphate 0.1 mol / L Tween 200.5% (v / v) Tris(2-carboxyethyl)phosphine 1 mmol / L Adjust the pH to 7.4 using hydrochloric acid or sodium hydroxide. Once the solution has completely wetted the membrane material, let it stand at room temperature for 30 minutes, and then dry it at 37°C to obtain the blood filtration membrane.

[0034] Fabrication of the reaction membrane: Biodyne C 0.45μm nylon membranes from Stofa were cut into 20cm × 0.8cm pieces and immersed in the following reaction membrane treatment solution for 10 minutes: HEPES 0.1 mol / L L-serine 10 mmol / L Cystathion β-synthase 50 U / mL Cystathion β-lyase 50 U / mL Pyridoxal phosphate 0.001% (w / v) Pyruvate oxidase 100 U / mL Flavin adenine dinucleotide 0.01% (w / v) Thiamine pyrophosphate 0.02% (w / v) Peroxidase 1000 U / mL 4-Aminoantipyrine 10 mmol / L MAOS 10 mmol / L Trehalose 2% (w / v) Potassium chloride 5 mmol / L Tween 200.5% (v / v) BSA 2% (w / v) pH=7.5 Then remove it and dry it at 37°C to obtain the reaction membrane.

[0035] Take two sheets of material, one as the top layer and the other as the bottom layer. Remove the release paper from the top layer and attach the diffusion layer. Ensure the diffusion layer is parallel to the longitudinal direction of the top layer, completely covering the sample well, and that both its top and bottom ends extend equidistant from the sample well. Figure 3 As shown.

[0036] Apply the reaction membrane to the bottom layer using the same method. Then place the blood filtration membrane on top of the reaction membrane, ensuring it is completely parallel to the reaction membrane and that its upper and lower ends extend equidistantly beyond the reaction membrane. Next, attach the upper layer with the diffusion membrane to this bottom layer, ensuring that the sample application wells and detection wells of the upper bottom layer are completely aligned. The diffusion membrane, blood filtration membrane, and reaction membrane are sandwiched between the upper and lower layers, resulting in the large detection device plate.

[0037] The large plate is cut into strips 10mm wide and 8cm long along its length to obtain the detection device 10.

[0038] Example 2 Biodyne C 0.45μm nylon film from Topvan was cut into 20cm × 0.8cm pieces and immersed in the following solution for 10 minutes: A 0.001% (w / v) ethanol solution of 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetic acid ester (CAS No.: 23973-20-8).

[0039] Then dry at 37°C, and then immerse this nylon membrane in the reaction membrane treatment solution in Example 1 for 10 minutes, and dry it again at 37°C to obtain the reaction membrane.

[0040] Everything else is the same as in Example 1.

[0041] Example 3 Biodyne C 0.45μm nylon film from Topvan was cut into 20cm × 0.8cm pieces and immersed in the following solution for 10 minutes: A 0.005% (w / v) ethanol solution of 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetate (CAS No.: 23973-20-8).

[0042] Then dry at 37°C, and then immerse this nylon membrane in the reaction membrane treatment solution in Example 1 for 10 minutes, and dry it again at 37°C to obtain the reaction membrane.

[0043] Everything else is the same as in Example 1.

[0044] Example 4 Biodyne C 0.45μm nylon film from Topvan was cut into 20cm × 0.8cm pieces and immersed in the following solution for 10 minutes: A 0.003% (w / v) ethanol solution of 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetate (CAS No.: 23973-20-8).

[0045] Then dry at 37°C.

[0046] 3-chloropyridine[2,3-b]pyrazine (CAS No.: 155535-23-2) was introduced into the reaction membrane treatment solution formulation of Example 1 to a concentration of 0.01% (w / v). The other components and their concentrations in the reaction membrane treatment solution remained unchanged from those in Example 1. The nylon membrane treated in the previous step was soaked in this reaction membrane treatment solution for 10 min and then dried at 37°C to obtain the reaction membrane.

[0047] Everything else is the same as in Example 1.

[0048] Example 5 Biodyne C 0.45μm nylon film from Topvan was cut into 20cm × 0.8cm pieces and immersed in the following solution for 10 minutes: A 0.004% (w / v) ethanol solution of 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetate (CAS No.: 23973-20-8).

[0049] Then dry at 37°C.

[0050] 7-Bromo-8-methylpyrido[2,3-b]pyrazine (CAS No.: 116605-69-7) was introduced into the reaction membrane treatment solution formulation of Example 1 to a concentration of 0.03% (w / v). The other components and their concentrations in the reaction membrane treatment solution remained unchanged from the example. The nylon membrane treated in the previous step was immersed in this reaction membrane treatment solution for 10 min and then dried at 37°C to obtain the reaction membrane.

[0051] Everything else is the same as in Example 1.

[0052] Example 6 Biodyne C 0.45μm nylon film from Topvan was cut into 20cm × 0.8cm pieces and immersed in the following solution for 10 minutes: A 0.004% (w / v) ethanol solution of 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetate (CAS No.: 23973-20-8).

[0053] Then dry at 37°C.

[0054] 7-Bromo-8-methylpyrido[2,3-b]pyrazine (CAS No.: 116605-69-7) and 2,3-dichloro-pyrido[2,3-b]pyrazine (CAS No.: 25710-18-3) were introduced into the reaction membrane treatment solution formulation of Example 1, with concentrations of 0.02% (w / v) and 0.05% (w / v), respectively. The other components and their concentrations in the reaction membrane treatment solution remained unchanged from those in Example 1. The nylon membrane treated in the previous step was immersed in this reaction membrane treatment solution for 10 min and then dried at 37°C to obtain the reaction membrane.

[0055] Everything else is the same as in Example 1.

[0056] Example 7 Biodyne C 0.45μm nylon film from Topvan was cut into 20cm × 0.8cm pieces and immersed in the following solution for 10 minutes: A 0.002% (w / v) ethanol solution of 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetate (CAS No.: 23973-20-8).

[0057] Then dry at 37°C.

[0058] The reaction membrane treatment solution of Example 1 was formulated with 3-chloropyridin[2,3-b]pyrazine (CAS No.: 155535-23-2), 7-bromo-8-methylpyrido[2,3-b]pyrazine (CAS No.: 116605-69-7), and 2,3-dichloropyrido[2,3-b]pyrazine (CAS No.: 25710-18-3) at concentrations of 0.04% (w / v), 0.01% (w / v), and 0.05% (w / v), respectively. The other components and their concentrations in the reaction membrane treatment solution remained unchanged from those in Example 1. The nylon membrane treated in the previous step was immersed in this reaction membrane treatment solution for 10 min and then dried at 37°C to obtain the reaction membrane.

[0059] Everything else is the same as in Example 1.

[0060] Example 8 The sensitivity of the detection in each embodiment was examined.

[0061] Preparation of stock solution (i.e., homocysteine ​​concentration of 0 μmol / L): BSA 5% (w / v) NaCl 0.9% (w / v) Then, homocysteine ​​solutions with concentrations of 0, 5, 10, 25, and 50 μmol / L were prepared using the mother liquor.

[0062] Add 30 μL of solution to the sample well of the detection device. After 5 minutes, read the reflectance of the reaction film at 630 nm from the detection well. The results are shown in the table below: Table 1: As can be seen from the table above, when detecting 0 μmol / L homocysteine ​​(i.e., reagent blank value), Example 1 showed the lowest reflectance (meaning its color was the deepest compared to other examples), and at the low value of 5 μmol / L, it was completely indistinguishable from the blank, and even higher. The 10 μmol / L sample was also very indistinguishable from the blank sample, indicating that the low-value sample was strongly interfered with by non-specific background color development, greatly affecting the accuracy of the detection.

[0063] In Examples 2 and 3, where 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetic acid ester was added, the reagent blank values ​​increased, indicating that non-specific color development was suppressed. Furthermore, in Example 2, the reflectance of the 50 μmol / L sample reached 0.425, and the color depth significantly exceeded that of Example 1 (0.510), indicating that the depth of the detection reaction was enhanced while suppressing background color development.

[0064] Examples 4 to 7, which added pyrido[2,3-b]pyrazine derivatives to 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetate, showed a continued increase in blank values, indicating that the non-specific colorimetric problem was further improved, and the depth of the colorimetric reaction was further enhanced. Overall detection performance was further improved.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. The technical features of the above embodiments can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification.

[0066] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; within the framework of this application, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of different aspects of one or more embodiments of this application as described above, which are not provided in detail for the sake of brevity.

[0067] One or more embodiments in this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of one or more embodiments in this application should be included within the protection scope of this application.

Claims

1. A homocysteine ​​detection device, characterized in that... The membrane comprises an upper layer, a diffusion layer, a blood filtration membrane, a reaction membrane, and a bottom layer. The upper layer has a sample application well for adding samples, and the bottom layer has a detection well for detection. The reaction membrane contains L-serine, cystathionine β-synthetase, cystathionine β-lyase, pyruvate oxidase, thiamine pyrophosphate, peroxidase, and a colorimetric reagent. The reaction membrane also contains 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetate.

2. The homocysteine ​​detection device according to claim 1, characterized in that... The mass concentration of the 3',4,4',5,5',6-hexahydroxy-3,6'-oxydi[tetrahydro-2H-pyran-2-methanol]octaacetic acid ester is 0.

001. 0.005%.

3. The homocysteine ​​detection device according to claim 2, characterized in that... The reaction membrane also contains pyrido[2,3-b]pyrazine derivatives.

4. The homocysteine ​​detection device according to claim 3, characterized in that... The pyrido[2,3-b]pyrazine derivatives are 3-chloropyrido[2,3-b]pyrazine, 7-bromo-8-methylpyrido[2,3-b]pyrazine, and 2,3-dichloropyrido[2,3-b]pyrazine.

5. The homocysteine ​​detection device according to claim 4, characterized in that... The mass concentration of the pyrido[2,3-b]pyrazine derivative is 0.01%. 0.05%.

6. The homocysteine ​​detection device according to claim 5, characterized in that... The blood filtration membrane contains tris(2-carboxyethyl)phosphine (TCEP).

7. The homocysteine ​​detection device according to claim 6, characterized in that... The colorimetric reagent is sodium N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline and 4-aminoantipyrine.

8. The homocysteine ​​detection device according to claim 7, characterized in that... The reaction membrane contains pyridoxal phosphate and flavin adenine dinucleotide.

9. The homocysteine ​​detection device according to claim 8, characterized in that... The blood filtration membrane is Topfan 1660.

10. The homocysteine ​​detection device according to claim 9, characterized in that... The reaction membrane is a Biodyne C 0.45μm nylon membrane.

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