A device and method for purifying urine protein

Abstract

The present invention discloses a device for purifying urine protein, comprising a filter tube, a filter adsorption sheet and a collection tube, wherein the collection tube is arranged at the liquid outlet of the filter tube, the filter adsorption sheet is arranged in the filter tube, and the filter adsorption sheet comprises a stacked nitrocellulose membrane and a glass fiber pad. A filter adsorption sheet is arranged between the filter tube and the collection tube, and the filter adsorption sheet can filter precipitated protein and adsorb soluble small molecule peptides, greatly simplifying the pre-treatment of clinical biological samples, facilitating the purification of proteins in samples, and reducing the difficulty of operation.

Description

Technical Field

[0001] The present invention relates to the field of medical detection technology, and in particular to a device and method for purifying urine protein. Background Art

[0002] Urine is a filtered product of blood. It has many unique advantages, such as non-invasive and easy collection, large-scale availability, relatively stable properties, and easy preservation, making it an ideal sample for clinical disease detection and research. Some studies have shown that under normal human physiological conditions, about 30% of the protein in urine is plasma protein, while the other 70% is composed of kidney and urogenital tract derived proteins. Therefore, urine can directly reflect the functional state of the kidneys and urinary system, and to a certain extent, it can reflect the functional state of the entire body. Therefore, urine is an important source for finding disease biomarkers. Biomarkers are molecules that reflect the normal or abnormal biological state of the body, including proteins, nucleic acids, small molecules, ions, etc., which can be used for the diagnosis and treatment evaluation of clinical diseases.

[0003] At present, the methods for detecting biomarkers in urine are very limited in clinical practice, and the sensitivity and specificity of the methods cannot meet the needs of clinical disease diagnosis. For example, the detection of urine protein usually uses chemical methods that cannot be classified, while the electrophoresis method can be classified, but the operation is cumbersome, there are many interference factors, and the sensitivity is not enough; the detection of small molecule compounds (such as dopamine, vitamins, amino acids, etc.) mostly uses capillary electrophoresis, immunological methods, etc., and there are many interference factors in the experiment, and the data fluctuates greatly. In recent years, with the development of proteomics technology, especially the development of proteomics technology and metabolomics technology based on mass spectrometry and chromatography, more and more potential biomarkers have been discovered. These biomarkers are helpful for auxiliary diagnosis, etiology analysis, evaluation of treatment effects, and optimization of treatment. However, for the detection of clinical biological samples based on mass spectrometry and chromatography, sample pretreatment is very critical, such as purification (or removal) of proteins in samples, removal of interfering substances, removal of ions, etc., especially the purification of proteins in samples, the related operations are cumbersome and difficult. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a device and method for purifying urine protein which is easy to operate.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a device for purifying urine protein, comprising a filter tube, a filter adsorption sheet and a collecting tube, the collecting tube is provided at the liquid outlet of the filter tube, the filter adsorption sheet is arranged in the filter tube, and the filter adsorption sheet comprises a stacked nitrocellulose membrane and a glass fiber liner.

[0006] In order to solve the above technical problems, the present invention also adopts a technical solution: a method for purifying urine protein, using the above-mentioned device for purifying urine protein for purification, comprising the following steps:

[0007] S1, preparing a urine protein precipitation reagent and a urine protein precipitation re-solution, wherein the urine protein precipitation reagent is kept cold for later use;

[0008] S2, fully mixing the sample and the urine protein precipitation reagent to form a mixed solution and then letting it stand;

[0009] S3, adding the mixed solution after standing in step S2 into the filter element tube and centrifuging the mixed solution;

[0010] S4, after the mixed solution in step S3 is centrifuged, the solution in the collection tube is collected to form a first sample;

[0011] S5. The first sample in step S4 can be subjected to metabolomics small molecule substance detection.

[0012] The beneficial effects of the present invention are as follows: a filter adsorption sheet is provided between the filter core tube and the collection tube, and the filter adsorption sheet can filter precipitated proteins and adsorb soluble small molecule peptides, which greatly simplifies the pretreatment of clinical biological samples, facilitates the purification of proteins in the samples, and reduces the difficulty of operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram of the structure of a device for purifying urine protein according to Embodiment 1 of the present invention;

[0014] Figure 2 This is a flow chart of a method for purifying urine protein according to Example 1 of the present invention;

[0015] Figure 3 This is an example diagram of a protein molecular weight standard according to Example 1 of the present invention;

[0016] Figure 4 This is a comparison diagram of the sample urine protein before and after SDS-PAGE gel electrophoresis treatment;

[0017] Figure 5 This is a comparison diagram of another sample of urine protein before and after SDS-PAGE gel electrophoresis treatment.

[0018] Description of labels:

[0019] 1. Filter tube;

[0020] 2. Filter adsorption sheet;

[0021] 3. Collection tube;

[0022] 4. Shell. DETAILED DESCRIPTION

[0023] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in combination with the implementation modes and the accompanying drawings.

[0024] The most critical concept of the present invention is that the filter adsorption sheet can filter precipitated proteins and adsorb soluble small molecule peptides, greatly simplifying the pre-treatment of clinical biological samples.

[0025] Please refer to Figures 1 to 5 A device for purifying urine protein comprises a filter tube, a filter adsorption sheet and a collecting tube, wherein the collecting tube is arranged at the liquid outlet of the filter tube, the filter adsorption sheet is arranged in the filter tube, and the filter adsorption sheet comprises a stacked nitrocellulose membrane and a glass fiber liner.

[0026] The structural principle of the present invention is briefly described as follows: the filter adsorption sheet can filter precipitated proteins and adsorb soluble small molecule peptides, separate the precipitated proteins and soluble small molecule peptides in the sample from the sample, and facilitate the separation of the precipitated proteins and soluble small molecule peptides from other components in the sample for separate study.

[0027] From the above description, it can be seen that the beneficial effects of the present invention are: a filter adsorption sheet is provided between the filter element tube and the collection tube, and the filter adsorption sheet can filter the precipitated protein and adsorb soluble small molecule peptides, which greatly simplifies the pretreatment of clinical biological samples, facilitates the purification of proteins in the samples, and reduces the difficulty of operation.

[0028] Furthermore, the pore size of the nitrocellulose membrane is 0.45 μm, and the thickness of the glass fiber pad is 1.5 mm.

[0029] Furthermore, the filter adsorption sheet 2 is arranged close to the liquid outlet.

[0030] It can be seen from the above description that the filter adsorption sheet arranged near the liquid outlet can effectively distinguish the filtered sample from the unfiltered sample, which is convenient for collection and use.

[0031] Furthermore, it also includes a shell 4, and the filter tube 1 and the collecting tube 3 are both arranged in the shell 4.

[0032] It can be seen from the above description that the filter tube and the collecting tube are both arranged in the outer shell, and the outer shell plays a protective role for the filter tube and the collecting tube.

[0033] A method for purifying urine protein, using the above-mentioned device for purifying urine protein for purification, comprises the following steps:

[0034] S1, preparing a urine protein precipitation reagent and a urine protein precipitation reconstitution solution, wherein the urine protein precipitation reagent is kept cold for later use;

[0035] S2, fully mixing the sample and the urine protein precipitation reagent to form a mixed solution and then letting it stand;

[0036] S3, adding the mixed solution after standing in step S2 into the filter tube 1 and centrifuging the mixed solution;

[0037] S4, after the mixed solution in step S3 is centrifuged, the solution in the collection tube 3 is collected to form a first sample;

[0038] S5. The first sample in step S4 can be subjected to metabolomics small molecule substance detection.

[0039] Furthermore, the method further includes S31 after step S3, wherein the filter tube 1 is replaced with another collection tube 3, and the urine precipitate protein re-solution is added to the filter tube 1 and mixed thoroughly, and the mixture is allowed to stand for 5 minutes;

[0040] S32, centrifuging the filter tube 1 after standing in step S31, collecting the solution in the tube 3 after centrifugation as the second sample, which can be subjected to proteomics analysis.

[0041] Furthermore, the urine protein precipitation reagent is 90% acetone, and the urine precipitated protein resolution is prepared from Tris-HCl and formic acid.

[0042] Furthermore, in step S2, the mixing ratio of the sample to the urine protein precipitation reagent is 1:4.

[0043] Furthermore, the cold storage temperature in step S1 is -20°C.

[0044] From the above description, it can be seen that storage in a low temperature environment is conducive to the stability of urine protein precipitation.

[0045] Embodiment 1

[0046] Please refer to Figures 1 to 5 Embodiment 1 of the present invention is: a device for purifying urine protein, comprising a filter tube 1, a filter adsorption sheet 2 and a collection tube 3, wherein the collection tube 3 is provided at the liquid outlet of the filter tube 1, the filter adsorption sheet 2 is arranged in the filter tube 1, and the filter adsorption sheet 2 comprises a stacked nitrocellulose membrane and a glass fiber liner.

[0047] Optionally, the pore size of the nitrocellulose membrane is 0.45 μm, and the thickness of the glass fiber pad is 1.5 mm.

[0048] Please refer to Figure 1 The filter adsorption sheet 2 is arranged close to the liquid outlet.

[0049] In detail, it also includes a shell 4, and the filter tube 1 and the collecting tube 3 are both arranged in the shell 4. In this embodiment, an upper cover is also provided at the opening of the shell 4, and the upper cover covers the top opening of the shell 4.

[0050] Please refer to Figure 2 This embodiment also relates to a method for purifying urine protein, which is performed using the above-mentioned urine protein purification device, comprising the following steps:

[0051] S1. Prepare a urine protein precipitation reagent and a urine protein precipitation reconstitution solution, wherein the urine protein precipitation reagent is stored cold for standby use, and optionally, the urine protein precipitation reagent is stored cold at a temperature of -20°C;

[0052] S2. Fully mix the sample and the urine protein precipitation reagent to form a mixed solution and then let it stand; preferably, the standing time is 10 minutes;

[0053] S3, adding the mixed solution after standing in step S2 to the filter element tube 1 and centrifuging the mixed solution, preferably, the standing time is ten minutes;

[0054] S4, after the mixed solution in step S3 is centrifuged, solution 3 in the collection tube is collected to form a first sample;

[0055] S5. The first sample in step S4 can be subjected to metabolomics small molecule substance detection.

[0056] Optionally, the method further includes S31 after step S3, wherein the filter tube 1 is replaced with another collecting tube 3, and the urine precipitated protein re-solution is added to the filter tube 1 and mixed thoroughly, and the mixture is allowed to stand for 5 minutes; S32, the filter tube after standing in step S31 is centrifuged, and the solution in the collecting tube 3 after centrifugation is the second sample, which can be subjected to proteomic analysis, and the protein can be further analyzed and diagnosed through proteomic analysis.

[0057] In this embodiment, the urine protein precipitation reagent is 90% acetone, the urine precipitated protein reconstitution solution is prepared by Tris-HCl and formic acid, the urine protein precipitation reagent is pre-cooled at -20°C for standby use, and the urine precipitated protein reconstitution solution is stored at room temperature.

[0058] Optionally, the specification of Tris-HCl is 0.1M, pH 7.4, and the formic acid concentration in the urine precipitate protein re-solution is 30%.

[0059] In detail, in step S2, the mixing ratio of the sample to the urine protein precipitation reagent is 1:4.

[0060] In detail, the thorough mixing in step S2 and step S31 can be mixed by blowing and mixing with a pipette gun; the centrifugal speed in step S3 and step S32 is 12000 rpm / min, the centrifugal treatment time is five minutes, and the ambient temperature during centrifugation is 4°C.

[0061] Please refer to Figure 3 , Figure 3The molecular weights of proteins from top to bottom in the example diagram are: 220KD, 135KD, 90KD, 66KD, 45KD, 35KD, 29KD, 20KD, 14KD. Figure 4 and Figure 5 , Figure 4 and Figure 5 The left side is the effect picture of the sample urine protein before SDS-PAGE gel electrophoresis treatment. Figure 4 On the right is the effect picture of the sample urine protein after SDS-PAGE gel electrophoresis treatment. Through obvious comparison, it can be seen that the effect picture of the urine after treatment has removed most of the protein interference. The sample can be repeatedly processed to further remove excess interfering proteins until satisfactory, which is convenient for subsequent analysis and research.

[0062] In summary, in the device for purifying urine protein provided by the present invention, a filter adsorption sheet is provided between the filter core tube and the collection tube, and the filter adsorption sheet can filter the precipitated protein and adsorb soluble small molecule peptides, which greatly simplifies the pretreatment of clinical biological samples, facilitates the purification of proteins in the samples, and reduces the difficulty of operation.

[0063] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for purifying urine protein, using a device for purifying urine protein for purification, characterized in that: The device for purifying urine protein comprises a housing, a filter tube, a filter adsorption sheet and a collection tube, wherein the filter tube and the collection tube are both arranged in the housing, the collection tube is arranged at the liquid outlet of the filter tube, the filter adsorption sheet is arranged in the filter tube, the filter adsorption sheet is arranged near the liquid outlet, and the filter adsorption sheet comprises a nitrocellulose membrane and a glass fiber liner arranged in layers; The following steps are involved: S1, preparing a urine protein precipitation reagent and a urine protein precipitation reconstitution solution, wherein the urine protein precipitation reagent is 90% acetone, and the urine protein precipitation reconstitution solution is prepared by Tris-HCl and formic acid, wherein the urine protein precipitation reagent is stored cold for later use; S2, fully mixing the sample and the urine protein precipitation reagent to form a mixed solution and then letting it stand; S3, adding the mixed solution after standing in step S2 into the filter element tube and centrifuging the mixed solution; S31, replace the filter tube with another collection tube, add the urine precipitate protein re-solution into the filter tube, mix thoroughly, and let it stand for 5 minutes; S32, centrifuging the filter tube after standing in step S31, collecting the solution in the tube after centrifugation as the second sample, and performing proteomic analysis; S4, after the mixed solution in step S3 is centrifuged, the solution in the collection tube is collected to form a first sample; S5. Performing metabolomics small molecule substance detection on the first sample in step S4.

2. The method for purifying urine protein according to claim 1, characterized in that: In step S2, the mixing ratio of the sample to the urine protein precipitation reagent is 1:

4.

3. The method for purifying urine protein according to claim 1, characterized in that: The cold storage temperature in step S1 is -20°C.

Images