CN209485830U - A device for enriching urine extracellular vesicles that can be used in hospitals and households - Google Patents

Abstract

The device of the utility model is applicable to the field of medical technology. It is a device for enriching extracellular vesicles in urine that can be used in hospitals and households. It does not need a centrifuge and other auxiliary equipment other than the device, and can be used in the early stage of chronic diseases. A non-invasive liquid biopsy device for diagnosis, the device includes: a sample pool, arranged at the upper end of the device, and the sample pool holds a urine sample to be filtered; a nano-filter, including at least one filter layer and extracellular vesicle collection device; the extracellular vesicle collector is arranged at the lower end of the filter layer, and the extracellular vesicle collector collects the urine extracellular vesicles; the nanofilter waste liquid collector has a urine impurity collection chamber ; the vacuum suction interface on the side wall of the waste liquid collector; a negative pressure component, which sucks between the waste liquid collector and the nano-filter to a preset negative pressure. Thereby, the device of the utility model realizes convenient and efficient extraction of urine extracellular vesicles.

Description

A device for enriching urine extracellular vesicles that can be used in hospitals and households

technical field

The utility model relates to the technical field of medical devices, in particular to a device for enriching urine extracellular vesicles that can be used by hospitals and families, and is used for non-invasive liquid biopsy for early diagnosis of chronic diseases.

Background technique

Urine, as the most commonly used detection of specific biomarkers for different diseases, is used to diagnose different diseases as a source of samples. Screening for accepted shortcomings. However, there are a large number of interferences of physiological proteins (such as Tamm-Hosfall protein) and other high-abundance components in urine, which makes it difficult for us to detect low-abundance but diagnostically valuable urine components that reflect the degree of disease changes. Although proteins, lipids, nucleic acids and other components in urine EVs extracellular vesicles (Extracelluar Vesicles, EVs) can be used as biomarkers to identify different diseases, most of them are also low-abundance components, and it is urgent to develop an enrichment The device can significantly improve the detection rate of EVs low-abundance components by enriching EVs. The utility model belongs to the technical field of biotechnology and clinical examination, and in particular relates to a device for early diagnosis of various chronic diseases that can be used in families and communities to enrich urine extracellular vesicles with a nano-membrane.

Extracellular vesicles are a type of membranous vesicles that originate from the endosome system and are secreted by cell polyvesicles through plasma membrane fusion, with a diameter of 30-1000nm. EVs contain a variety of proteins, lipids, various A variety of RNA (ribonucleic acid, ribonucleic acid) components, etc., can be secreted by many different types of cells, widely distributed in various body fluids, can be used as carriers between cells, and transmit various physiological and pathological information through the cargo carrier function. Functions such as intracellular signal transduction, immune regulation, establishment of tumor escape mechanism, and mediation of regeneration and degeneration processes are new targets for clinical diagnosis and treatment with high development potential. The secretion of extracellular vesicles and their The internal components such as protein, lipid, RNA, etc. can change, and these changes are related to the pathogenesis of different diseases. The detection of the secretion of extracellular vesicles and the differences in the internal components can be used for the differential diagnosis of different diseases.

Urine, as the most commonly used detection of specific biomarkers for different diseases, is used to diagnose different diseases as a source of samples. Screening for accepted shortcomings.

The existing technical solutions include the following:

(1) Ultracentrifugation, using different ultracentrifugation speeds or density gradient centrifugation to separate and extract sample EVs, is an expensive EVs separation and extraction method that is currently only used in scientific research institutes. Although the purity of EVs obtained by ultracentrifugation is high, it is only suitable for the processing of a small number of samples due to the need for an expensive ultracentrifuge and corresponding consumables, and the entire extraction process is time-consuming.

(2) Relying on the magnetic bead adsorption method of immunoaffinity capture, the magnetic beads containing specific antibodies are used to incubate with the sample to react with the EVs containing the corresponding antigen on the membrane surface of the sample, and then the magnetic beads-EVs are combined by magnetic adsorption body separated. Although the extraction and separation of specific EVs by magnetic bead adsorption method has high purity and can distinguish subtypes, it requires special processing of magnetic beads, consumes a lot of reagent consumables, and has low yield and yield.

(3) Co-precipitation-based kit extraction methods, commercial kits such as Exo-Quick, Exo-Spin, etc., use the principle of polymerization co-precipitation, adding reagents to the sample to inhibit the hydration of EVs, making it easy to precipitate, The pellet of EVs was then enriched by subsequent low-speed centrifugation. The disadvantage of the kit-based extraction method based on co-precipitation is that impurities such as aggregated particles, lipoproteins, and RNA complexes will be produced, and the purity of the obtained EVs is low, and the kit is expensive and cannot be used in clinical practice on a large scale.

In summary, the actual use of the existing technology requires professional technicians and professional equipment that are not available in ordinary hospitals, and cannot be promoted in most hospitals, let alone for home use. Therefore, it is necessary to develop a simple and easy-to-use new technical device.

Utility model content

In view of the above-mentioned defects, the purpose of this utility model is to provide a device for enriching urine extracellular vesicles that can be used by hospitals and families, so as to realize the enrichment of urine extracellular vesicles without centrifuges and other auxiliary equipment other than this device. The extraction of vesicles can be used in hospitals and at home.

In order to achieve the above purpose, the utility model provides a device for enriching urine extracellular vesicles that can be used by hospitals and families, including:

The sample pool is arranged on the upper end of the device, and the sample pool holds the urine sample to be filtered;

The nano filter is arranged between the sample pool and the waste liquid collector, including at least one filter layer and an extracellular vesicle collector; the extracellular vesicle collector is arranged at the lower end of the filter layer, and the cells an extracellular vesicle collector collects the urine extracellular vesicles;

The nano-filter waste liquid collector has a urine impurity collection cavity, and the waste liquid collector is sleeved on the outside of the nano-filter; the vacuum suction port on the side wall of the waste liquid collector;

The negative pressure component is connected to the vacuum suction interface, and suctions the space between the waste liquid collector and the nano filter to a preset negative pressure.

According to the described device, the filter layer comprises:

The initial filter layer includes a filter membrane support frame and an initial filter membrane, and the initial filter membrane is embedded on the filter membrane support frame;

The nanofiltration layer includes a nanomembrane support frame and a nanofiltration membrane; the nanomembrane support frame is embedded in the bottom of the nanofilter and connected to the side wall of the nanofilter; the extracellular vesicle collector It is arranged at the center of the nanofiltration layer, and the extracellular vesicle collector is detachably connected with the nanomembrane support frame.

According to the device, the vacuum suction interface is arranged on the upper part of the side wall of the waste liquid collector, and the vacuum suction interface is used to connect the negative pressure component;

The top of the device has a support edge, and the support edge and the top nozzle of the waste liquid collector are in a closed structure;

The device also includes a cap, which is arranged on the top of the sample pool, and is detachably connected with the outer wall of the waste liquid collector or detachably connected with the edge of the support.

According to the device, the filter membrane support frame includes a plurality of first support bars, one ends of the plurality of first support bars are respectively connected to the bottom of the side wall of the sample pool, and the other ends are connected together ;

The nano-membrane support frame includes a plurality of second support bars, one end of the plurality of second support bars is respectively connected to the bottom of the side wall of the nano-filter, and the other ends of the plurality of second support bars are connected together.

According to the device, the initial filter membrane has a plurality of first filter holes, and the size of the first filter hole aperture is 2 microns; the nanofiltration membrane has a plurality of second filter holes, and a plurality of the first filter holes 2. The size of the filter pore diameter is 20 nanometers;

The capacity of the extracellular vesicle collector is 5-20 milliliters.

According to the device, the initial filter layer is an initial filter layer for filtering urine impurities including cell debris and larger than extracellular vesicles; Extracellular vesicles, and the nanofiltration membrane layer that filters urine impurities including soluble proteins and the like; the urine extracellular vesicles obtained by filtering through the initial filtration layer and the nanofiltration membrane layer are collected in the cell in the outer vesicle collector.

According to the device, the longitudinal section of the primary filter layer is V-shaped or arc-shaped; the longitudinal section of the nano-filtration layer is V-shaped or arc-shaped.

According to the device, the shape of the sample pool, the nano-filter and the waste liquid collector are all cylindrical;

The outer walls of the sample pool, the nanofilter and the waste liquid collector are made of transparent material;

The bottom of the waste liquid collector is detachably connected to the outer wall of the waste liquid collector.

According to the device, scale lines are provided on the outer walls of the sample pool, the nanofilter, and the waste liquid collector, and these scale lines are not on the same straight line in the lateral direction;

The inner wall of the bottom of the waste liquid collector and the outer wall of the waste liquid collector are respectively provided with corresponding inner and outer threads.

According to described device, described device also comprises:

A rinse cup for containing ultrapure water is arranged outside the waste liquid collector, the material of the rinse cup is transparent material, and the rinse cup is provided with a scale line, and the capacity of the rinse cup is 200 ml;

A pipette for blowing the retentate on the nanofiltration membrane, arranged outside the body of the waste liquid collector; and

The negative pressure component is a vacuum pump.

The utility model sets the device for enriching urine extracellular vesicles that can be used in hospitals and families to include a sample pool, a nano-filter and a waste liquid collector. The bottom of the nano-filter is provided with a nano-membrane, and the nano-filter A trap chamber rich in EVs nanometers is formed inside, and a waste liquid collection chamber for urine impurities is formed between the interior of the waste liquid collector and the bottom of the nano-filter. The nozzle at the top of the device is a closed structure, and the side wall of the waste liquid collector is provided with a vacuum pump suction pipe interface, which is connected to the vacuum pump through the suction pipe. In this way, the vacuum pump can be used to carry out negative pressure suction, and the urine specimen can be filtered by using the hydrostatic pressure and the pressure on both sides of the filter membrane to obtain EVs, and there is no need for complicated operations, centrifuges and other experimental instruments and reagents other than this device. Processing, operation is simple and quick. The device is easy to carry, adopts consumable materials with low cost, and is suitable for large-scale popularization and use in families and community hospitals.

Description of drawings

Fig. 1 is a schematic structural diagram of a device for enriching urine extracellular vesicles that can be used by hospitals and families provided by the embodiment of the present invention;

Fig. 2 is a schematic structural diagram of a device for enriching urine extracellular vesicles that can be used by hospitals and families provided by the embodiment of the present invention;

Fig. 3 is a schematic structural diagram of the device for enriching urine extracellular vesicles shown in Fig. 2 that can be used by both hospitals and families;

Fig. 4A is a schematic diagram of the structure of the filter membrane support in the device for enriching urine extracellular vesicles that can be used by hospitals and families provided by the embodiment of the present invention;

Fig. 4B is a schematic structural view of the filter membrane support frame in the device for enriching urine extracellular vesicles that can be used by hospitals and families provided by the embodiment of the present invention;

Fig. 5A is a schematic diagram of the structure of the nanomembrane support frame in the device for enriching urine extracellular vesicles that can be used by hospitals and families provided by the embodiment of the present invention;

Fig. 5B is a schematic diagram of the structure of the nanomembrane support frame in the device for enriching urine extracellular vesicles that can be used by hospitals and households provided by the embodiment of the present invention;

Fig. 6 is a schematic diagram of the test results of enriching urine extracellular vesicles by the device for enriching urine extracellular vesicles provided by the embodiment of the present invention, which can be used in hospitals and households.

Detailed ways

In order to make the purpose, technical solution and advantages of the utility model clearer, the utility model will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the utility model, and are not intended to limit the utility model.

Referring to Figures 1 to 3, in one embodiment of the present invention, a device 100 for enriching urine extracellular vesicles that can be used by both hospitals and families is provided, including:

A sample pool 10 is arranged on the upper end of the device, and the sample pool 10 holds the urine sample to be filtered;

The nano filter 20 is arranged between the sample pool 10 and the waste liquid collector 30, including at least one filter layer and an extracellular vesicle collector 23; the filter layer is connected to the extracellular vesicle collector 23 , the extracellular vesicle collector 23 is arranged at the lower end of the filter layer, and the extracellular vesicle collector 23 collects the urine extracellular vesicles;

The waste liquid collector 30 has a urine impurity collection cavity, and the waste liquid collector 30 is sleeved on the outside of the nanofilter 20; the vacuum suction port 315 on the side wall of the waste liquid collector 30;

The negative pressure component is connected to the vacuum suction interface 315 to suck the space between the waste liquid collector 30 and the nano filter 20 to a preset negative pressure.

In this embodiment, the device 100 for enriching urine extracellular vesicles that can be used in hospitals and households includes a sample pool 10 , a nanofilter 20 and a waste liquid collector 30 . Wherein the sample pool 10 is used to place the urine sample to be filtered; the nanofilter 20 includes at least one filter layer and an extracellular vesicle collector 23, the filter layer has a filter membrane, which can filter out the urine in the urine sample impurities, the urine extracellular vesicles are extracted and stored in the extracellular vesicle collector 23 . The device 100 for enriching urine extracellular vesicles that can be used in hospitals and families is low in cost, simple in operation, and short in time. The yield and purity of extracellular vesicles obtained are high and their structure is complete. Other instruments and equipment can be used for large-scale promotion in families and communities. The device 100 for enriching urine extracellular vesicles that can be used in hospitals and homes can be used in homes and communities, and can be used for further detection and analysis of early diagnosis of various chronic diseases by extracting extracellular vesicles. Change the preset negative pressure to achieve the purpose of enrichment.

Referring to Figures 1 to 3, in one embodiment of the present invention, the filter layer 21 includes:

The initial filter layer 21 includes a filter membrane support frame 211 and an initial filter membrane 222; the filter membrane support frame 211 is connected to the bottom of the sample pool 10, and the initial filter membrane 222 is embedded in the filter membrane support frame 211;

The nanofiltration layer 22 includes a nanomembrane support frame 212 and a nanofiltration membrane 213; the nanomembrane support frame 212 is arranged at the bottom of the nanofilter 20 and is connected with the sidewall of the nanofilter 20; the nanofiltration membrane 213 is embedded in the nanomembrane On the support frame 212; the extracellular vesicle collector 23 is arranged at the center of the nanofiltration layer 22; and the extracellular vesicle collector 23 is detachably connected with the nanomembrane support frame 212, as in the extracellular vesicle collector 23 The top is provided with an elastic clip, and the center of the nanomembrane support frame 212 is provided with a clip corresponding to the elastic clip, so that the extracellular vesicle collector 23 can be installed and taken out conveniently. The bottom of the sample pool 10 is a filter membrane support frame 211 inclined to the middle, and the inside of the filter membrane support frame 211 is embedded with an initial filter membrane 222; the bottom of the nanofilter 20 is a nano membrane support frame 212 inclined to the middle, and the nano membrane support frame The inside of 212 is embedded with a nanofiltration membrane 213, and the center of the nanofilter 20 forms a V-shaped conical EVs collector 23 for extracellular vesicles; the outer wall of the nanofilter 20 and the waste liquid collector 30 of the vacuum chamber is provided with a volume scale.

In addition, the upper part of the side wall of the waste liquid collector 30 is provided with a vacuum suction interface 315, and the vacuum suction interface 315 is used to connect a vacuum pump; The nozzle is a closed structure. The edges of the initial filter membrane 222 and the nanofiltration membrane 213 have a closed structure, and the nozzle at the top of the nanofilter 20 is provided with a supporting edge 32, which is a closed structure between the supporting edge 32 and the nozzle at the top of the waste liquid collector 30, and the vacuum chamber The nozzle at the top of the waste liquid collector 30 is provided with a cap, and the outer side of the upper half of the pipe wall is provided with a vacuum suction interface 315 connected to a negative pressure component. By docking, the space between the waste liquid collector 30 and the nano-filter 20 can be sucked to a preset negative pressure to increase the filtration efficiency. In addition, the device 100 for enriching urine extracellular vesicles, which can be used in hospitals and households, also includes a cap 50, which is placed on the top of the sample pool 10, and is detachably connected to the outer wall of the waste liquid collector 30 or connected to The support edge 32 is detachably connected. For example, it can be screwed to the outer wall of the waste liquid collector 30 , or snapped to the support edge 32 , and a sealing rubber pad matching the support edge 32 is provided on the inner side of the cap 50 . Thus, the device 100 for enriching urine extracellular vesicles, which can be used in both hospitals and households, can be sealed to form a vacuum environment. When it is necessary to enrich urine extracellular vesicles using the device 100 for enriching urine extracellular vesicles that can be used in hospitals and households, after adding the sample solution, rotate the cap 50 with the outer wall of the waste liquid collector 30 Tightly, after the enrichment is completed, unscrew the cap 50 to clean the device 100 and so on. Fig. 3 is a schematic diagram of the structure of the device for enriching urine extracellular vesicles shown in Fig. 2, which can be used by both hospitals and households, without a cap. In addition, referring to FIG. 2 and FIG. 3 , the bottom 301 of the waste liquid collector is detachably connected to the outer wall of the waste liquid collector 30 . The two can be connected through a suitable clamping piece or a threaded connection. Specifically, the inner wall of the bottom 301 of the waste liquid collector and the outer wall of the waste liquid collector 30 are respectively provided with corresponding internal and external threads. After the enrichment process is completed, the enriched urine extracellular vesicles can be taken out by removing the bottom 301 of the waste liquid collector, and then removing the extracellular vesicle collector 23 .

Referring to Fig. 4A and Fig. 4B, in one embodiment of the present utility model, the filter membrane support frame 211 includes a plurality of first support bars 2111, and one end of the plurality of first support bars 2111 is connected to the sample pool 10 respectively. The connection of the side wall of the bottom, its other end is connected together; Thus, the initial filter layer 21 has a certain inclination, can filter very well; The connection will form a conical shape, as shown in Figure 4A, or a circular arc shape, as shown in Figure 4B.

Referring to Fig. 5A and Fig. 5B, described nanomembrane support frame 212 comprises a plurality of second support strips 2121, and one end of a plurality of second support strips 2121 is respectively connected with the bottom of the sidewall of nanofilter 20, and its other end is connected at Together. A plurality of second support strips 2121 are connected to the bottom of the side wall of the nanofilter 20 to form a cone shape, or an arc shape, as shown in FIG. 5A, or an arc shape, as shown in FIG. 5B.

Specifically, the initial filter membrane 222 has a plurality of first filter holes, and the diameter of the first filter hole is 2 microns; the nanofiltration membrane 213 has a plurality of second filter holes, and the diameter of a plurality of the second filter holes is The size is 20 nanometers; the capacity of the extracellular vesicle collector 23 is 5-20 milliliters. Preferably, the initial filter layer 21 is an initial filter layer that filters cell debris and other urine impurities that are larger than extracellular vesicles; Nanofiltration layer of extracellular vesicles for urinary impurities. The urine extracellular vesicles obtained by filtering through the initial filter layer 21 and the nanofiltration membrane layer 22 are collected in the extracellular vesicle collector 23 . The connection between the extracellular vesicle collector 23 and the bottom of the nanomembrane support frame 212 may be connected to the common connection ends of the plurality of second support bars 2121, and the connection ends are ring-shaped to facilitate the dripping of urine extracellular vesicles. Fall into extracellular vesicle collector 23.

In one embodiment of the present utility model, the sample is first poured into the sample pool 10, and the vacuum pump suction pipe interface on the outside of the waste liquid collector 30 in the vacuum chamber is connected to the vacuum pump for negative pressure suction. Lateral pressure difference, the sample passes through the initial filter membrane 222 to remove cell debris and other urine impurities that are larger than extracellular vesicles; then the sample reaches the nanofilter 20, and when passing through the nanofilter 213, the interception diameter can be 30 to 1000 nanometers EVs; the final enriched EVs will be collected into the V-shaped EVs collector (extracellular vesicle collector 23), that is, to complete the enrichment of urine extracellular vesicles. The utility model has the advantages of low cost, simple operation, short time consumption, high yield and purity of obtained extracellular vesicles and complete structure, no need for centrifuges and other instruments and equipment other than the device, and can be popularized on a large scale for family and community use, etc. .

In one embodiment of the present utility model, the longitudinal section of the primary filter layer 21 is V-shaped or arc-shaped; the longitudinal section of the nano-filtration layer 22 is V-shaped or arc-shaped. The shape of the sample pool 10, the nanofilter 20 and the waste liquid collector 30 are all cylindrical; the outer walls of the sample pool 10, the nanofilter 20 and the waste liquid collector 30 are made of transparent materials. Scale lines are provided on the outer walls of the sample pool 10 , the nanofilter 20 and the waste liquid collector 30 , and these scale lines are not on the same straight line in the lateral direction. Thus, the scales on the outer walls of the sample pool 10 , the nanofilter 20 and the waste liquid collector 30 , as well as the capacity of the solutions currently loaded in each container can be clearly seen.

In addition, the device 100 for enriching urine extracellular vesicles that can be used by both hospitals and families also includes:

A rinse cup for containing ultrapure water is arranged outside the waste liquid collector 30. The material of the rinse cup is a transparent material, and the rinse cup is provided with a scale mark. The capacity of the rinse cup is 200ml;

A pipette for blowing the retained liquid on the nanofiltration membrane 213 is arranged outside the waste liquid collector 30 .

The specific method for enriching urine extracellular vesicles by using the device 100 for enriching urine extracellular vesicles that can be used in hospitals and households provided by the utility model is as follows:

After the samples are collected, they are added to the sample pool in batches according to the volume of 10 volumes of the sample pool. The vacuum suction interface 315 outside the waste liquid collector 30 of the vacuum chamber is connected to a vacuum pump for negative pressure suction, and the hydrostatic pressure and the pressure difference on both sides of the filter membrane are used , the sample passes through the initial filter membrane 222 with a pore size of 2 microns to remove cell debris and other urine impurities larger than extracellular vesicles. When the sample reaches the nanofilter 20, EVs with a diameter of 30 to 1000 nanometers can be intercepted when passing through the nanofiltration membrane 213 with a pore size of 20 nanometers, while the remaining soluble proteins, etc. seep out from the nanofiltration membrane 213 and enter the vacuum chamber waste liquid Collector 30. When about 10ml of the retained liquid in the nanofilter 20 remains, add 200ml of ultrapure water into the EVs nano-concentrator with a rinse cup, and continue the above nanomembrane process. When the remaining 5ml of the retentate in the nanofilter 20 remains, blow the retentate with a pipette to mix it evenly and collect it into the extracellular vesicle collector 23 of EVs. The retentate is the final urine extracellular vesicle solution. Then the bottom of the waste liquid collector 30 can be disassembled, and finally the extracellular vesicle collector 23 can be taken out to obtain the final urine extracellular vesicle solution for detection of chronic diseases, such as kidney disease and diabetes.

(1) Filtration with an initial filter membrane with a pore size of 2 microns: take 30ml of urine samples from healthy volunteers for research, pour the urine samples into the sample pool first, and connect the vacuum pump suction tube outside the vacuum chamber waste liquid collector The upper vacuum pump is used for negative pressure pumping, using the hydrostatic pressure and the pressure difference on both sides of the filter membrane, the sample passes through the initial filter membrane to remove cell debris and other urine impurities that are larger than extracellular vesicles;

(2) Nanofiltration membrane filtration with a pore size of 20 nanometers: the sample reaches the nanofiltration membrane, and EVs with a diameter of 30 to 1000 nanometers can be intercepted through the nanofiltration membrane;

(3) Enrichment of EVs: When the retentate in the nanofilter remains about 10ml, add 200ml of ultrapure water into the EVs nanoconcentrator with a rinse cup, and continue the above nanomembrane process. When the remaining 5ml of the retentate in the nanofilter remained, pipette the retentate to mix it evenly and collect it into the extracellular vesicle collector of EVs. The retentate was the final urine extracellular vesicle solution. Finally, the enriched EVs will be collected into the V-shaped EVs collector (extracellular vesicle collector), that is, to complete the enrichment of urinary extracellular vesicles.

The primary filter membrane and nanofiltration membrane can be purchased from Spectrum LabsInc of Spectrum (Shanghai) Trading Co., Ltd. The primary filter membrane with a pore size of 2 microns removes cell debris and other urine impurities that are larger than extracellular vesicles. Nano The filter membrane has a molecular weight cut-off of 1000KDa and EVs with a cut-off diameter of 30 to 1000 nanometers.

(4) Transmission electron microscope observation: Take 20ul of urine extracellular vesicles prepared in step (3), spot on the copper grid, and after 5 minutes, negatively stain with 3% (W/V) phosphotungstic acid for 2 minutes, super Washed twice with pure water, tested after drying for 5 minutes, adjusted the focal length of the transmission electron microscope, and observed the shape and particle size of urine extracellular vesicles. The specific results are shown in Figure 6. It can be seen from Figure 6 that urine extracellular The vesicles are cup-shaped or round, with a particle size of 30-1000nm.

(5) NanoSight detection: dilute urine extracellular vesicles with PBS, equipotentially dilute to 200 times, load the sample into the syringe, 2-3ml each time, clean the detection tank with ultrapure water before and after loading the sample, and adjust the NanoSight Instrument parameters, to the appropriate focal length, for analysis and recording of extracellular vesicles. The main peak of the particle size of urine extracellular vesicles was 102nm, and the sample was relatively pure.

(6) Test results

Under the transmission electron microscope, the extracellular vesicles of urine are cup-shaped or spherical and evenly distributed, as shown in Figure 6.

The average size of urine extracellular vesicles was 102nm.

Compared with the existing commonly used ultracentrifugation method, magnetic bead adsorption method and kit extraction method, the extracellular vesicles provided by the utility model have the following advantages: extracellular vesicles with the same purity as the above method can be enriched without the need for Centrifuge and other instruments and equipment other than the device, because the nanofiltration membrane 213 with a pore size of 20 nanometers in the device 100 can retain EVs with a diameter of 30 to 1000 nanometers, while the rest of the soluble proteins, etc., seep from the nanofiltration membrane 213 out. The device 100 can process a large amount of urine samples at one time without complicated operations. The device 100 can be used in a laboratory equipped with a small vacuum pump, or even at home, without the need for a centrifuge and other experimental instruments and reagents other than the device. The device 100 uses a vacuum pump to draw air under negative pressure, and utilizes hydrostatic pressure and the pressure difference between the two sides of the filter membrane to realize it, and the operation is simple and fast. The device 100 is easy to carry, uses consumables with low cost, and is suitable for large-scale popularization and use in families and community hospitals. Wherein the initial filter membrane 222 and the nanofiltration membrane 213 are respectively made of microporous membranes with corresponding pore sizes, for example, the nanofiltration membrane 213 is made of a nanomembrane with a pore size of 20 nm, and the molecular weight cut-off of the nanomembrane is 1000 kDa.

The utility model sets the device for enriching urine extracellular vesicles that can be used in hospitals and families to include a sample pool, a nano-filter and a waste liquid collector. The bottom of the nano-filter is provided with a nano-membrane, and the nano-filter A trap chamber rich in EVs nanometers is formed inside, and a waste liquid collection chamber for urine impurities is formed between the interior of the waste liquid collector and the bottom of the nano-filter. The nozzle at the top of the collector is a closed structure, and the outer side of the upper half of the pipe wall is provided with a vacuum pump suction pipe interface, which is connected to the vacuum pump through the suction pipe. In this way, the vacuum pump can be used to carry out negative pressure suction, and the urine specimen can be filtered by using the hydrostatic pressure and the pressure on both sides of the filter membrane to obtain EVs, and there is no need for complicated operations, centrifuges and other experimental instruments and reagents other than this device. Processing, operation is simple and quick. The device is easy to carry, adopts consumable materials with low cost, and is suitable for large-scale popularization and use in families and community hospitals.

Of course, the utility model can also have other various embodiments, and those skilled in the art can make various corresponding changes and deformations according to the utility model without departing from the spirit and essence of the utility model, but These corresponding changes and deformations should all belong to the protection scope of the appended claims of the present utility model.

Claims (10)

1. the device of a kind of hospital and the home-use extracellular vesica of enrichment urine that can make characterized by comprising

Sample pool, is set to the upper end of described device, and the sample pool holds the urine specimen to be filtered；

Nanofilter is set between the sample pool and liquid waste collector, including at least one filter layer and cell external capsule Steep collector；The extracellular vesica collector is set to the filter layer lower end, and the extracellular vesica collector collects institute State the extracellular vesica of urine；

Nanofilter liquid waste collector has urine impurity collecting chamber, and the liquid waste collector is socketed on the nanometer The outside of filter；Vacuum suction interface on the liquid waste collector side wall；

Negative pressure component is connected to the vacuum suction interface, will aspirate between the liquid waste collector and the nanofilter For preset negative pressure.

2. the device of hospital according to claim 1 and the home-use extracellular vesica of enrichment urine that can make, feature It is, the filter layer includes:

Inceptive filtering layer, including filter membrane support frame and initial filter membrane, the initial filter membrane are embedded on filter membrane support frame；

Nanofiltration layer, including nanometer film support frame and nanofiltration membrane；The nanometer film support frame is embedded in the nanofiltration The bottom of device is connect with the side wall of the nanofilter；The extracellular vesica collector is set to the nanofiltration layer Centre, and the extracellular vesica collector is detachably connected with the nanometer film support frame.

3. the device of hospital according to claim 2 and the home-use extracellular vesica of enrichment urine that can make, feature It is, the vacuum suction interface is set to the side wall upper part of the liquid waste collector, and the vacuum suction interface is for connecting Negative pressure component；

The top of described device has support edge, and the support is enclosed construction along the top nozzle with the liquid waste collector；

Described device further includes nut cap, is covered on the top of the sample pool, detachable with the lateral wall of the liquid waste collector Connection is detachably connected with the support edge.

4. the device of hospital according to claim 2 and the home-use extracellular vesica of enrichment urine that can make, feature Be, the filter membrane support frame includes multiple first support items, one end of multiple first support items respectively with the sample pool The bottom of side wall connects, and the other end links together；

The nanometer film support frame includes multiple second support items, one end of multiple second support items respectively with the nanofiltration The bottom of the side wall of device connects, and the other end links together.

5. the device of hospital according to claim 2 and the home-use extracellular vesica of enrichment urine that can make, feature It is, the initial filter membrane has multiple first filter hole, and the size in first filter hole aperture is 2 microns；The nanometer Filter membrane has multiple second filter hole, and the size in multiple second filter hole apertures is 20 nanometers；

The capacity of the extracellular vesica collector is 5~20 milliliters.

6. the device of hospital according to claim 5 and the home-use extracellular vesica of enrichment urine that can make, feature It is, the inceptive filtering layer is the inceptive filtering layer that filtering includes cell fragment and the urine impurity greater than extracellular vesica； The nanofiltration layer is the extracellular vesica of urine that retention is greater than nanometer film filtration aperture, and filtering includes soluble protein The nanofiltration layer of urine impurity；The urine cell obtained by the inceptive filtering layer and nanofiltration layer filtering Outer vesica is collected in the extracellular vesica collector.

7. the device of hospital according to claim 2 and the home-use extracellular vesica of enrichment urine that can make, feature It is, the longitudinal cross-section of the inceptive filtering layer is V-shape or circular arc type；The longitudinal cross-section of the nanofiltration layer is V word Type or circular arc type.

8. the device of hospital according to claim 1 and the home-use extracellular vesica of enrichment urine that can make, feature It is, the shape of the sample pool, nanofilter and the liquid waste collector is in cylindrical；

The outer wall of the sample pool, nanofilter and the liquid waste collector is made of transparent material；

The bottom of the liquid waste collector and the outer wall of the liquid waste collector are detachably connected.

9. the device of hospital according to claim 8 and the home-use extracellular vesica of enrichment urine that can make, feature It is, graduation mark, and these institutes is provided on the outer wall of the sample pool, nanofilter and the liquid waste collector It states on the same straight line of graduation mark not in a lateral direction；

The outer wall of the inner wall of the bottom of the liquid waste collector and the liquid waste collector is respectively arranged with corresponding inside and outside spiral shell Line.

10. the device of hospital according to claim 3 and the home-use extracellular vesica of enrichment urine that can make, feature It is, described device further include:

For holding the washing cup of ultrapure water, it is set to that the liquid waste collector is external, the material of the washing cup is transparent material Material, and graduation mark is provided on the washing cup, the capacity of the washing cup is 200 milliliters；

For blowing and beating the pipettor of the trapped fluid in the nanofiltration membrane, it is external to be set to the liquid waste collector；And

The negative pressure component is vacuum pump.