Full-automatic urine cell efficient enrichment system

Abstract

The utility model relates to a full-automatic urine cell efficient enrichment system. Comprising a urine cell separation module, a sample module, a sample introduction module, a liquid storage module, a waste extraction module and a circuit control module, the urine cell separation module comprises an upper cover plate, a microporous filter membrane and a lower cover plate; the upper cover plate is provided with a sample phase inlet, a cross flow layer chamber and a liquid storage phase outlet. According to the utility model, not only is a circulating laminar flow filtering system adopted, but also circuit control is adopted, and laminar flow filtering can be continuously circulated according to a set program, so that the tumor cells in the urine can be fully automatically, efficiently and circularly enriched.

Description

Technical Field

[0001] The utility model relates to the technical field of medical detection equipment, in particular to a full-automatic urine cell efficient enrichment system. Background Art

[0002] Cancer cells in urine are of great significance for the diagnosis, treatment, and monitoring of cancer. Detecting cancer cells in urine offers numerous advantages. Urine is a non-invasive biological specimen that is easily accessible and painless to the patient, making it suitable for cancer screening and monitoring. Furthermore, cancer cells, along with cell fragments, DNA, RNA, and proteins, found in urine can provide important diagnostic markers, aiding in the diagnosis and monitoring of early-stage cancers. Finally, the detection of disseminated tumor cells in urine may indicate the presence of a primary tumor, recurrence, or metastasis, facilitating early therapeutic intervention. Cancer cells and biomarkers in urine can more accurately represent pathological changes within the urinary system, playing a particularly important role in monitoring urinary tumors such as bladder cancer. Therefore, the enrichment of cancer cells in urine, particularly bladder cancer cells, is an important research topic. Current methods for enriching bladder cancer cells in urine primarily include cell sorting, urine pretreatment, and biomarker detection. Cell sorting technologies include immunomagnetic bead-based methods, microfluidics, and microbubble-based methods, which can quickly and efficiently isolate cancer cells from complex urine samples. Urine pretreatment technologies include ultrasound, centrifugation, and microporous membrane methods, which are used to remove particulate matter from urine and extract cancer cells. Biomarker detection technologies enrich cancer cells by detecting specific marker molecules in urine, such as DNA, RNA, or proteins.

[0003] However, existing methods for enriching bladder cancer cells in urine still have some shortcomings. For example, existing technologies may cause damage or inactivation of cancer cells, have low enrichment efficiency, are complex to operate, and require a high sample size. In addition, some methods may also have a certain enrichment effect on other cells or components in urine, leading to interference or misjudgment. Therefore, there is an urgent need to optimize the technology for enriching bladder cancer cells in urine to improve its accuracy, sensitivity, and specificity for better application in cancer diagnosis, treatment, and monitoring. Utility Model Content

[0004] In order to solve the above problems, the utility model proposes a fully automatic urine cell efficient enrichment system.

[0005] The utility model provides a fully automatic urine cell efficient enrichment system, comprising: a urine cell separation module, a sample module, a sampling module, a liquid storage module, a waste extraction module, and a circuit control module; the urine cell separation module comprises an upper cover, a microporous filter membrane, and a lower cover; the upper cover is provided with a sample phase inlet, a cross-flow layer chamber, and a liquid storage phase outlet; the lower cover is provided with a tangential flow layer chamber and a waste liquid phase outlet; the microporous filter membrane is located between the upper cover and the lower cover; after the urine in the sample module enters the urine cell separation module through the sampling module, the waste liquid that passes through the microporous filter membrane enters the waste extraction module, and the urine that does not pass through the microporous filter membrane enters the sample module again after passing through the liquid storage module; the sample module comprises a liquid storage port and a sampling port; the circuit control module is electrically connected to the sampling module, the liquid storage module, and the waste extraction module.

[0006] Furthermore, the liquid storage port of the sample module is located above the sample module; and the sample inlet is located at the bottom of the sample module.

[0007] Furthermore, the sampling module includes a sampling pipeline and a one-way stop valve. One end of the sampling pipeline is connected to the sampling port of the sample module through the one-way stop valve, and the other end is connected to the sample inlet of the urine cell separation module.

[0008] Furthermore, the liquid storage module includes a liquid storage peristaltic pump and a liquid storage pipeline; one end of the liquid storage pipeline is connected to the liquid storage phase outlet of the urine cell separation module through the liquid storage peristaltic pump, and the other end is connected to the liquid storage port of the sample module.

[0009] Furthermore, the waste extraction module includes a waste extraction peristaltic pump, a waste extraction pipeline and a waste liquid barrel. One end of the waste extraction pipeline is connected to the waste liquid outlet of the urine cell separation module through the waste extraction peristaltic pump, and the other end is connected to the waste liquid barrel.

[0010] Furthermore, a detection sensor is provided on the waste extraction pipeline near the waste liquid phase outlet to detect whether there is any waste liquid in the waste extraction pipeline.

[0011] Furthermore, when enriching bladder cancer cells, the pore size of the microporous filtration membrane is 8-10 μm.

[0012] The beneficial effects of the utility model are as follows:

[0013] The urine cell separation module of the present invention adopts a laminar flow filtration system, which is invented based on the principles of microporous membrane filtration and microfluidics. Laminar flow filtration is a process in which the water phase flows parallel to the surface of the microporous membrane, forming a large pressure on the surface of the microporous membrane, causing cells to enter the filter pores for filtration and screening, thereby achieving the effect of enrichment. Circulating laminar flow filtration is a type of tangential flow filtration, which requires pressure to push the filtered solution through the filter pores of the microporous membrane. Since the pushing speed is very fast and the liquid flow rate is very rapid, it will not clog the filter pores of the microporous membrane. Bladder cancer cells are generally between 20μm and 100μm in size. By setting the pore size of the microporous filter membrane, target cells can be enriched. For example, when enriching bladder cancer cells, the pore size of the microporous filter membrane is 8-10μm. Bladder cancer cells can hardly pass through the microporous membrane, while non-target cells and crystalline impurities can be filtered out through the micropores. Ultimately, bladder cancer cells in urine can be enriched efficiently and with high sensitivity. The utility model not only adopts a circulating laminar flow filtration system, but also adopts circuit control, which can continuously circulate laminar flow filtration according to a set program, thereby realizing fully automatic and efficient circulatory enrichment of tumor cells in urine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 , a schematic diagram of the structure of the fully automatic urine cell efficient enrichment system of Example 1;

[0015] Figure 2 , an exploded diagram of the urine cell separation module structure of Example 1;

[0016] Figure 3 , schematic diagram of laminar flow filtration achieved by the urine cell separation module of Example 1;

[0017] Figure 4 , a comparison chart of laminar flow filtration and dead-end filtration in Example 2;

[0018] Figure 5 , micrograph of cells on the microporous filtration membrane of Example 3;

[0019] Figure 6 , in situ hybridization image of bladder cancer cells enriched on the microporous filtration membrane of Example 4;

[0020] Figure 7 , Comparison chart of bladder cancer patient detection in Example 4. DETAILED DESCRIPTION

[0021] The present invention will be further described below with reference to the following embodiments.

[0022] Example 1: A fully automatic urine cell efficient enrichment system:

[0023] like Figure 1-2As shown, the fully automatic urine cell efficient enrichment system of the present invention includes a urine cell separation module 1, a sample module 2, a sampling module, a liquid storage module, a waste extraction module, and a circuit control module; the urine cell separation module 1 includes an upper cover plate 14, a microporous filter membrane 16 and a lower cover plate 15; the upper cover plate 14 is provided with a sample phase inlet 11, a cross-flow layer chamber, and a liquid storage phase outlet 12; the lower cover plate 15 is provided with a tangential flow layer chamber and a waste liquid phase outlet 13; the microporous filter membrane 16 is located between the upper cover plate and the lower cover plate; after the urine in the sample module 2 enters the urine cell separation module 1 through the sampling module, the waste liquid that passes through the microporous filter membrane 16 enters the waste extraction module, and the urine that does not pass through the microporous filter membrane 16 enters the sample module 2 again after passing through the liquid storage module; the sample module 2 includes a liquid storage port and a sampling port; the circuit control module is electrically connected to the sampling module, the liquid storage module, and the waste extraction module. In this embodiment, the pore size of the microporous filtration membrane is 8-10 μm.

[0024] Specifically, the liquid storage port of the sample module 2 is located above the sample module; and the sample inlet is located at the bottom of the sample module.

[0025] Specifically, the sampling module includes a sampling pipeline and a one-way stop valve 7 . One end of the sampling pipeline is connected to the sampling port of the sample module 2 through the one-way stop valve 7 , and the other end is connected to the sample phase inlet 11 of the urine cell separation module 1 .

[0026] Specifically, the liquid storage module includes a liquid storage peristaltic pump 4 and a liquid storage pipeline; one end of the liquid storage pipeline is connected to the liquid storage phase outlet 12 of the urine cell separation module 1 through the liquid storage peristaltic pump 4, and the other end is connected to the liquid storage port of the sample module 2.

[0027] Specifically, the waste extraction module includes a waste extraction peristaltic pump 5, a waste extraction pipeline and a waste liquid barrel 3. One end of the waste extraction pipeline is connected to the waste liquid phase outlet 13 of the urine cell separation module 1 through the waste extraction peristaltic pump 5, and the other end is connected to the waste liquid barrel 3.

[0028] Specifically, a detection sensor 6 is provided on the waste extraction pipeline near the waste liquid phase outlet 13 for detecting whether there is waste liquid in the waste extraction pipeline. Once it is detected that there is no liquid in the waste extraction pipeline, waste extraction and reflux are automatically stopped.

[0029] When the system is working, the urine sample to be enriched is placed in the sample module 2, the pipeline interfaces are connected, the flow rate of the liquid storage peristaltic pump 4 is set to 2000μL / min, the flow rate of the waste extraction peristaltic pump 5 is set to 1000μL / min, and the two peristaltic pumps are started at the same time. The urine sample to be enriched quickly fills the upper cover plate cross-flow layer chamber and the lower cover plate tangential flow layer chamber of the urine cell separation module 1 through the pipeline, and the system operates normally. Figure 3The urine cell separation module realizes laminar flow filtration as shown in the schematic diagram.

[0030] After the enrichment is completed, the liquid storage peristaltic pump 4 is first turned off. After observing that the residual liquid is completely discharged from the waste liquid port, the waste extraction peristaltic pump 5 is turned off, and finally the microporous filter membrane 16 of the urine cell separation module 1 is taken out for further operation.

[0031] During operation, the detection sensor 6 always detects the waste extraction pipeline. Once it detects that there is no liquid in the waste extraction pipeline, it automatically stops waste extraction and reflux.

[0032] Example 2: Comparison between laminar flow filtration and dead-end filtration:

[0033] A 100 mL urine sample was collected and divided into two equal parts. Using the same microporous filter membrane with a pore size of 8-10 μm, one part was filtered and observed using the urine cell separation module described in Example 1, and the other part was filtered using conventional upper and lower dead-end filtration (dead-end filtration refers to a one-time filtration mode from the upper end to the lower end in a single direction). The flow rate of the waste liquid port was 1000 μL / min. After filtration, the microporous filter membrane was removed and observed under a bright field microscope. Figure 4 As shown in the figure, A represents the surface of the microporous filtration membrane using laminar flow filtration in the urine cell separation module, and B represents the surface of the microporous filtration membrane using dead-end filtration. It can be clearly seen from the figure that at the same microporous filtration membrane pore size of 8-10μm and flow rate, the surface of the microporous filtration membrane using dead-end filtration is significantly clogged with cells, while the surface of the microporous filtration membrane using laminar flow filtration is highly clean, indicating that it has a significant anti-cell stacking effect, thereby more effectively improving the purity of the enriched target cells.

[0034] Example 3, determination of bladder cancer cell enrichment efficiency:

[0035] To verify the system's efficiency in enriching bladder cancer cells in urine, a certain number of cultured human bladder transitional papilloma cells (RT4) were added to phosphate buffered saline (PBS) as target cells. After the system of Example 1 was run, the number of cells on the microporous filter membrane was observed, counted, and finally analyzed by statistics. Figure 5 Shown is a microscopic image of cells on the microporous filtration membrane after operation of the system, indicating that the system can effectively retain bladder cancer cells.

[0036] Table 1 System determination of RT4 cell retention efficiency in Example 1

[0037]

[0038] As shown in Table 1, the overall recovery rate of this system is higher than 80% when different cell numbers (4 groups, with cell numbers of 5, 45, 105, and 405, respectively, and 3 replicates in each group) are added, indicating that the system has a high enrichment efficiency for samples containing different cell numbers, especially when the cell number is less than single digits, which almost reaches the level of rare cells, and the system still has a good capture and enrichment efficiency.

[0039] Example 4: In situ hybridization experiment of cells enriched by laminar flow filtration:

[0040] To verify its downstream analytical capabilities, we performed in situ hybridization on samples obtained by laminar flow filtration using the urine cell separation module described in Example 1 in Example 2. The specific processing steps are briefly described as follows:

[0041] 1. Wash 1: Add 200 μL of 2×SSC to the microporous filter membrane and wash twice, 5 minutes each time. After each wash, dry the filter paper.

[0042] 2. Digestion: Preheat bladder cancer probe enzyme working solution at 37°C. Preheat 10 mL of weak hydrochloric acid solution (0.01 M) in a 37°C water bath. Add 20 μL of pepsin (1%) before use and mix thoroughly (adjust the amount to suit your needs). Preheat in advance. For bladder cancer cell slides, digest for 5-10 minutes. Place the microporous membrane in a 37°C hybridization apparatus for digestion. Blot dry with filter paper.

[0043] 3. Wash 2: Wash with PBS for 3 minutes, blot dry with filter paper; wash with 1% polymethanol for 5-10 minutes, blot dry with filter paper; wash with PBS for 3 minutes, blot dry with filter paper; wash with 2×SSC for 5 minutes, blot dry with filter paper.

[0044] 4. Dehydration: 70%, 85%, and 100% ethanol, 100 μL each, for 1-2 min each. Blot dry with filter paper and set aside.

[0045] 5. Denaturation Hybridization (Darkroom Operation): The dual-color bladder cancer probes CEP3 / CEP7 and P16(9P21) / CEP17 must be removed in advance and allowed to equilibrate at room temperature for 5-10 minutes. Once the probes have re-thawed to room temperature, shake them upside down and briefly centrifuge. Place 10 μL of the probes onto the microporous membrane, cover with a coverslip (avoid air bubbles), seal the edges with rubber tape, and place the slide on a hybridizer. Denature at 83°C for 5 minutes, and hybridize at 42°C for 2-16 hours.

[0046] 6. Take out the hybridized slide, remove the rubber glue on the cover slip, gently peel off the cover slip with tweezers, and take out the microporous membrane.

[0047] 7. Place the microporous membrane at room temperature and wash with 2×SSC for 1 minute; then dry it with filter paper.

[0048] 8. Remove the plate and immerse it in preheated 68°C 0.3% NP-40 / 0.4×SSC for 2 minutes after hybridization;

[0049] Blot dry with filter paper and repeat twice.

[0050] 9. Wash with deionized water or 2×SSC for 1 minute and blot with filter paper.

[0051] 10. Drop 5 μL of DAPI counterstain onto the slide area, gently place the microporous membrane on the counterstain area, then drop 5 μL of DAPI counterstain onto the microporous membrane and cover with a coverslip.

[0052] 11. Observe under a fluorescence microscope.

[0053] like Figure 6 The image shows in situ hybridization of RT4 cells on a microporous membrane. Green fluorescent spots represent CEP3 and CEP17 probes, while red fluorescent spots represent CEP7 and P16 probes. The probe luminescence spots are bright and clear, with a low background. Furthermore, based on the characteristics of RT4 cells, CEP3 and CEP17 are abnormally amplified, and P16 is absent. The experimental results match the reference well.

[0054] Based on this process, patient samples (bladder cancer patients) were collected and enriched by laminar flow filtration using the fully automatic urine cell efficient enrichment system of Example 1, and then subjected to in situ hybridization testing. Figure 7 As shown, Figure 7 A is a diagram of cell hybridization test after laminar flow filtration enrichment. Figure 7 Figure B shows the pathology results of a conventional urine sediment scraping. It can be seen that cells enriched using the fully automated urine cell efficient enrichment system of Example 1 can produce pathology results that match those of conventional urine sediment scrapings. This indicates that the device, in clinical use, specifically in bladder cancer patients, has a very high degree of match with the gold standard pathology results, and therefore has great clinical and economic value.

[0055] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced within the present invention.

[0056] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A fully automatic urine cell efficient enrichment system, characterized by: include: A urine cell separation module (1), a sample module (2), a sample injection module, a liquid storage module, a waste extraction module, and a circuit control module; the urine cell separation module (1) comprises an upper cover plate (14), a microporous filter membrane (16), and a lower cover plate (15); the upper cover plate (14) is provided with a sample phase inlet (11), a transverse flow layer chamber, and a liquid storage phase outlet (12); the lower cover plate (15) is provided with a shear flow layer chamber and a waste liquid phase outlet (13); the microporous filter membrane (16) is provided with a sample phase inlet (11), a transverse flow layer chamber, and a liquid storage phase outlet (12); the lower cover plate (15) is provided with a shear flow layer chamber and a waste liquid phase outlet (13); the microporous filter membrane (16) is provided with a sample phase inlet (11), a transverse flow layer chamber, and a liquid storage phase outlet (12); the lower cover plate (15) is provided with a transverse ... ) is located between the upper cover plate and the lower cover plate; after the urine in the sample module (2) enters the urine cell separation module (1) through the sample injection module, the waste liquid that passes through the microporous filter membrane (16) enters the waste extraction module, and the urine that does not pass through the microporous filter membrane (16) enters the sample module (2) again after passing through the liquid storage module; the sample module (2) includes a liquid storage port and a sample injection port; the circuit control module is electrically connected to the sample injection module, the liquid storage module, and the waste extraction module.

2. The fully automatic urine cell efficient enrichment system according to claim 1, characterized in that: The liquid storage port of the sample module (2) is located above the sample module; and the sample inlet is located at the bottom of the sample module.

3. The fully automatic urine cell efficient enrichment system according to claim 1, characterized in that: The sampling module comprises a sampling pipeline and a one-way stop valve (7); one end of the sampling pipeline is connected to the sampling port of the sample module (2) through the one-way stop valve (7), and the other end is connected to the sample phase inlet (11) of the urine cell separation module (1).

4. The fully automatic urine cell efficient enrichment system according to claim 1, characterized in that: The liquid storage module comprises a liquid storage peristaltic pump (4) and a liquid storage pipeline; one end of the liquid storage pipeline is connected to the liquid storage phase outlet (12) of the urine cell separation module (1) through the liquid storage peristaltic pump (4), and the other end is connected to the liquid storage port of the sample module (2).

5. The fully automatic urine cell efficient enrichment system according to claim 1, characterized in that: The waste extraction module comprises a waste extraction peristaltic pump (5), a waste extraction pipeline and a waste liquid bucket (3); one end of the waste extraction pipeline is connected to the waste liquid phase outlet (13) of the urine cell separation module (1) through the waste extraction peristaltic pump (5), and the other end is connected to the waste liquid bucket (3).

6. The fully automatic urine cell efficient enrichment system according to claim 4, characterized in that: A detection sensor (6) is provided on the waste extraction pipeline near the waste liquid phase outlet (13) for detecting whether there is still waste liquid in the waste extraction pipeline.

7. The fully automatic urine cell efficient enrichment system according to claim 1, characterized in that: When enriching bladder cancer cells, the pore size of the microporous filtration membrane (16) is 8-10 μm.

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