A fully automatic circulating tumor cell enrichment device

Abstract

The present invention relates to the technical field of fully automatic detection of circulating tumor cells; specifically, it is a fully automatic circulating tumor cell enrichment device, comprising: a chip storage unit, a first reagent storage unit, a second reagent storage unit, a sensing device, a shaking device and a transfer device; the first reagent storage unit comprises a plurality of cleaning liquid storage boxes and at least one waste liquid storage box; the second reagent storage unit comprises a plurality of test reagent storage racks arranged in sequence; the shaking device is used to fully shake the liquid injected into the chip storage unit and accelerate the reaction; the chip storage unit comprises a structure for storing a positive separation circulating tumor cell chip; the device realizes the fully automatic capture and staining function of circulating tumor cells in blood samples, has the advantages of easy use and standardized process, can provide standardized sample processing means for various tumors, such as prostate tumors and colon cancer tumors, and has important significance for basic research and clinical application of tumors.

Description

Technical Field

[0001] The invention relates to the technical field of fully automatic detection of circulating tumor cells; in particular to a fully automatic circulating tumor cell enrichment device. Background Art

[0002] Circulating tumor cells (CTCs) refer to tumor cells that fall off from the primary tumor and enter the peripheral blood circulation. They have important clinical significance in the prognosis, efficacy prediction, efficacy evaluation, and research on recurrence, metastasis, and drug resistance mechanisms of tumor patients. As far as the current technological development is concerned, because of their extremely rare number in the internal circulation and high individual heterogeneity, their enrichment and detection are the biggest technical bottlenecks currently faced.

[0003] Currently, the commonly used enrichment methods are mainly based on physical properties or surface biochemical characteristics:

[0004] 1. Physical characteristics include size, density, mechanics, charge, etc. Mainly include: density gradient centrifugation, filtration, microfluidic chips based on fluid mechanics, etc. Physical enrichment methods generally have a higher flux, but the separation purity is often lower.

[0005] 2. Surface biochemical characteristics refer to the use of specific antibodies to specifically bind to cell surface antigens to enrich CTCs. It mainly includes: positive enrichment method and negative enrichment method. The negative enrichment method usually uses immunomagnetic beads and other methods to remove white blood cells and other blood cells, but there is a problem of low separation purity. The positive enrichment method has high separation purity, but it depends on surface antigens. For example, the EpCAM antigen-based method cannot capture cells after epithelial-mesenchymal transition (EMT).

[0006] It can be seen that although the positive enrichment method has the advantage of high separation purity, its reliance on surface antigens will inevitably require higher precision in operation. At present, the steps of capturing and staining circulating tumor cells in the positive enrichment method still require manual operation, resulting in problems such as low capture efficiency and low cell activity. Therefore, we propose a device for fully automatic capture and staining of circulating tumor cells. Summary of the invention

[0007] In order to solve the shortcomings of the existing treatment scheme, the present invention provides a fully automatic circulating tumor cell enrichment device, including a body of the device, a cover is arranged on the body; the body is provided with;

[0008] The chip storage unit includes a chip storage box; both ends of the chip storage box are rotatably connected to the lower part of the body; a plurality of chip storage pools are arranged inside the chip storage box;

[0009] A shaking device is provided under the machine body, and the shaking device comprises a driving motor, an eccentric wheel is provided on the driving motor, a connecting rod is provided on the surface of the eccentric wheel, and the connecting rod is hinged to the lower end of the chip storage box;

[0010] A first reagent storage unit, the first reagent storage unit comprising a plurality of cleaning liquid storage boxes and at least one waste liquid storage box; the cleaning liquid storage box is connected to the chip storage tank via a connection pipe, and a first driving device is arranged between the cleaning liquid storage box and the chip storage tank; the waste liquid storage box is connected to the lower end of the chip storage tank via a connection pipe, and a second driving device is arranged between the waste liquid storage box and the chip storage tank;

[0011] A second reagent storage unit, the second reagent storage unit comprising a plurality of test reagent storage racks arranged in sequence;

[0012] The transfer device includes a moving frame, the moving frame is slidably connected to the inside of the machine body, and one end of the moving frame away from the bottom wall of the machine body is located directly above the chip storage pool; the inside of the moving frame is connected to the second reagent storage unit through a hose; a third driving device is arranged between the moving frame and the second reagent storage unit;

[0013] The sensor device is located at the bottom of the cleaning liquid storage box and the end of the corresponding connecting pipe, and at the top of the waste liquid storage box and the front end of the corresponding connecting pipe; the sensor device is also located at the bottom of the test reagent storage rack and the end of the hose close to the mobile rack;

[0014] Furthermore, a slide groove is provided at one end of the chip storage pool away from the transfer rack, and the slide groove is located on the upper side of the chip storage pool. A cover plate is slidably connected inside the slide groove, and the cover plate closes the upper end of the chip storage pool in an initial state.

[0015] Furthermore, a triangular block is provided inside the chip storage pool, the triangular block is a right-angled triangular block, two right-angled sides of the triangular block are in contact with the inner wall of the chip storage pool, and the inclined surface of the triangular block is inclined toward the direction of the slide slot;

[0016] Two limiting belts are provided at one end of the cover plate located inside the chip storage pool, and the two limiting belts are located on both sides of the cover plate; two winding shafts are provided on the inner wall of the chip storage pool close to the slide slot, and when the cover plate is fully opened, the two winding shafts are respectively located directly below the two limiting belts, and the two limiting belts are respectively wound around the corresponding winding shafts.

[0017] Furthermore, two installation grooves are provided on both sides of the inclined surface of the triangular block, and the interior of the installation groove is slidably connected to a limit plate, and a push block is provided below the limit plate, and the lower side of the push block is arranged in an arc shape;

[0018] The inner wall of the installation groove is provided with a sliding groove, and the push block is slidably connected inside the sliding groove;

[0019] The triangular block has two adjustment slots disposed inside, the two adjustment slots are located at the lower sides of the two mounting slots respectively, and the lower end of the push block is located inside the adjustment slot; a slider is slidably connected to the inside of the adjustment slot through a spring, and one end of the slider close to the sliding slot is provided with an inclined surface;

[0020] A rectangular groove is formed at one end of the triangular block away from the slide groove, and an arc groove is formed inside the triangular block, one end of the arc groove is connected to the rectangular groove, and the other end is connected to the adjustment groove;

[0021] An arc block is slidably connected inside the arc groove, one end of the arc block is located inside the rectangular groove, and the other end is located inside the adjustment groove. A push plate is provided inside the rectangular groove, and the push plate is connected to the end of the arc block located inside the rectangular groove; the end of the arc block located inside the adjustment groove contacts the slider.

[0022] Furthermore, a rolling ball is arranged at the arc-shaped end surface at the lower end of the pushing block; when the sliding block moves, the rolling ball contacts the inclined surface on the sliding block.

[0023] Furthermore, the second reagent storage unit includes a refrigeration device to keep the temperature in the test reagent storage rack constant at 3-5°C;

[0024] Furthermore, the first driving device is a single-channel peristaltic pump, the second driving device is a multi-channel peristaltic pump, and the third driving device is a single-channel peristaltic pump.

[0025] Furthermore, the cleaning liquid storage box, the waste liquid storage box, and the second reagent storage unit are each independently a warehouse, a tube, a hole, and a groove structure.

[0026] The above technical solution of the present invention has the following advantages compared with the prior art:

[0027] 1. The device provided by the present invention realizes the function of fully automatic capture and staining of circulating tumor cells in blood samples, has the advantages of easy use and standardized process, and can provide standardized sample processing methods for prostate tumors, which is of great significance to the basic research and clinical application of prostate tumors.

[0028] 2. Compared with the internationally recognized CTC capture device Cellsearch sorting system, the device provided by the present invention is only one-eighth of its volume, and realizes the fully automatic capture and immunofluorescence staining of CTC cells. Combined with the subsequent automatic fluorescence microscopy imaging analysis, the optimization process of regression and repeatability results can be standardized. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0030] Figure 1 is a stereogram of the enrichment device of the present invention;

[0031] Figure 2 It is a stereogram of another viewing angle of the enrichment device in the present invention;

[0032] Figure 3 yes Figure 2 Front view of the

[0033] Figure 4 It is a view of the internal structure of the enrichment device of the present invention;

[0034] Figure 5 It is a structural view of the chip storage pool in the present invention;

[0035] Figure 6 It is a structural view of the limiting belt in the present invention;

[0036] Figure 7 It is a structural view of the sliding groove, the adjusting groove and the sliding block in the present invention.

[0037] In the figure; body 1, machine cover 11, chip storage unit 2, chip storage box 21, chip storage pool 22, slide groove 221, cover plate 222, triangular block 223, limiting belt 224, winding shaft 225, shaking device 3, driving motor 31, eccentric wheel 32, connecting rod 33, first reagent storage unit 4, cleaning liquid storage box 41, waste liquid storage box 42, connecting pipe 43, second reagent storage unit 5, test reagent storage rack 51, transfer device 52, mobile rack 53, hose 54, installation slot 6, limiting plate 61, pushing block 62, rolling ball 621, sliding slot 63, adjusting slot 64, slider 65, rectangular slot 66, arc slot 67, arc block 68, pushing plate 69. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in combination with specific implementation methods and with reference to the accompanying drawings; it should be understood that these descriptions are only exemplary and are not intended to limit the scope of the present invention; in addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present invention.

[0039] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting; thus, other examples of the exemplary embodiments may have different values.

[0040] Embodiment 1:

[0041] Figures 2 to 7As shown; a preferred embodiment, a fully automatic circulating tumor cell enrichment device, comprising a body 1 of the device, on which a cover 11 is provided; the body 1 is provided with;

[0042] The chip storage unit 2 includes a chip storage box 21; both ends of the chip storage box 21 are rotatably connected to the lower part of the body 1; a plurality of chip storage pools 22 are arranged inside the chip storage box 21;

[0043] A shaking device 3 is provided below the machine body 1, and the shaking device 3 comprises a driving motor 31, an eccentric wheel 32 is provided on the driving motor 31, a connecting rod 33 is provided on the surface of the eccentric wheel 32, and the connecting rod 33 is hinged to the lower end of the chip storage box 21;

[0044] A first reagent storage unit 4, the first reagent storage unit 4 includes a plurality of cleaning liquid storage boxes 41 and at least one waste liquid storage box 42; the cleaning liquid storage box 41 is connected to the chip storage tank 22 via a connection pipe 43, and a first driving device is provided between the cleaning liquid storage box 41 and the chip storage tank 22; the waste liquid storage box 42 is connected to the lower end of the chip storage tank 22 via a connection pipe 43, and a second driving device is provided between the waste liquid storage box 42 and the chip storage tank 22;

[0045] A second reagent storage unit 5, the second reagent storage unit 5 comprises a plurality of test reagent storage racks 51 arranged in sequence;

[0046] The transfer device 52 includes a moving frame 53, which is slidably connected to the inside of the body 1, and one end of the moving frame 53 away from the bottom wall of the body 1 is located directly above the chip storage pool 22; the inside of the moving frame 53 is connected to the second reagent storage unit 5 through a hose 54; a third driving device is provided between the moving frame 53 and the second reagent storage unit 5;

[0047] The sensor device is located at the bottom of the cleaning liquid storage box 41 and the end of the corresponding connecting tube 43, and at the top of the waste liquid storage box 42 and the front end of the corresponding connecting tube 43; the sensor device is also located at the bottom of the test reagent storage rack 51 and the end of the hose 54 close to the mobile rack 53;

[0048] The chip storage pool 22 is provided with a slide groove 221 at one end away from the transfer rack. The slide groove 221 is located at the upper side of the chip storage pool 22. A cover plate 222 is slidably connected inside the slide groove 221. The cover plate 222 initially closes the upper end of the chip storage pool 22.

[0049] The driving device includes a first driving device connected to the cleaning liquid storage box 41 to drive the cleaning liquid into the chip storage unit 2, a third driving device connected to the test reagent storage rack 51 to drive the test reagent into the chip storage unit 2, and a second driving device connected to the chip storage unit 2 to drive the waste liquid into the waste liquid storage box 42; the first driving device, the second driving device and the third driving device are used for the transfer of cleaning liquid, test reagent and waste liquid, so the fully automatic circulating tumor cell enrichment device can be equipped with a corresponding control program to control the operation of the transfer device 52 as needed;

[0050] In a preferred embodiment, the first reagent storage unit 4 includes a cleaning liquid storage box 41 and a waste liquid storage box 42, and each of the cleaning liquid storage box 41 is configured to store reagent tubes arranged in sequence; the cleaning liquid storage box 41 is used to store cleaning liquid, and the waste liquid storage box 42 is used to store waste liquid. The number of cleaning liquid storage boxes 41 can be selected according to actual conditions, and is not limited to one; the type of cleaning liquid is also selected according to actual conditions, for example, including PBS buffer, paraformaldehyde and Triton X-100, etc.;

[0051] When the chip storage pool 22 needs to be cleaned, the staff can control the button on the enrichment device, and the button controls the single-channel peristaltic pump through the controller to pump the cleaning liquid in the cleaning liquid storage box 41 into the chip storage pool 22. The sensor device is located at the bottom of the cleaning liquid storage box 41 and the connection between the corresponding connecting pipe 43 and the chip storage pool 22, so that the sensor device can sense the remaining amount of cleaning liquid in the cleaning liquid storage box 41 in real time, and completely pump the cleaning liquid in the connecting pipe 43 corresponding to the cleaning liquid storage box 41 into the chip storage pool 22; After the shaking device 3 is started, the driving motor 31 in the shaking device 3 is started. During the rotation of the driving motor 31, the eccentric wheel 32 is driven to rotate. During the rotation of the eccentric wheel 32, the connecting rod 33 on its surface is driven to work. One end of the connecting rod 33 is connected to the chip storage box 21. The two ends of the chip storage box 21 are hinged at the bottom of the machine body 1, so that when the connecting rod 33 moves, the chip storage box 21 is driven to shake, and the chip storage pool 22 set inside the chip storage box 21 is synchronously swung, so that the cleaning liquid inside the chip storage pool 22 swings, and the inside of the chip storage pool 22 is cleaned;

[0052] After the cleaning is completed, the multi-channel peristaltic pump of the second driving device is operated to extract the cleaned liquid in the chip storage unit 2, and the multi-channel peristaltic pump draws the cleaned waste liquid in the chip storage pool 22 into the waste liquid storage box 42 through the connecting pipe 43; wherein a sensor device is arranged at the top of the waste liquid storage box 42 and at the connection between the connecting pipe 43 of the waste liquid storage box 42 and the chip storage pool 22, so that after the waste liquid in the chip storage unit 2 is completely extracted and the waste liquid in the connecting pipe 43 corresponding to the waste liquid storage box 42 completely enters the waste liquid storage box 42, the multi-channel peristaltic pump stops working;

[0053] In a preferred embodiment, the second reagent storage unit 5 includes at least three test reagent storage racks 51, and the test reagent storage racks 51 are respectively placed with reagent tubes arranged in sequence, and the test reagent storage racks 51 are used to store test reagents, and the test reagents are immunofluorescence staining reagents for CTC staining, for example, including FITC-anti-CD45 test reagents, PE-anti-CK test reagents and DAPI test reagents;

[0054] In addition, the chip storage pool 22 in the chip storage unit 2 of the present invention is used to place the positive isolated circulating tumor cell chip;

[0055] When testing the positive isolated circulating tumor cell chip, first after cleaning the chip storage pool 22, the staff slides the cover plate 222 away from the slide groove 221 in the chip storage pool 22, and then places the positive isolated circulating tumor cell chip into the chip storage pool 22, and then controls the single-channel peristaltic pump of the third drive device to work, and the single-channel peristaltic pump of the third drive device works to pump the test reagent in the reagent tube placed in the test reagent storage rack 51 into the chip storage pool 22; when the test reagent in one of the chip storage pools 22 is submerged in the positive isolated circulating tumor cell, the test reagent in the reagent tube placed in the test reagent storage rack 51 is pumped into the chip storage pool 22; when the test reagent in one of the chip storage pools 22 is submerged in the positive isolated circulating tumor cell, the test reagent in the reagent tube placed in the test reagent storage rack 51 is pumped into the chip storage pool 22; when the test reagent in one of the chip storage pools 22 is submerged in the positive isolated circulating tumor cell, the test reagent in the reagent tube placed in the test reagent storage rack 51 is pumped into the chip storage pool 22; when the test reagent in the chip storage pool ... After the cell chip, the mobile frame 53 in the transfer device 52 moves on the body 1 (the moving method of the mobile frame 53 is the slide rail device in the prior art and will not be described in detail here), and moves to the upper end of other chip storage pools 21 until the test reagents are injected into all chip storage pools 21; after the reagent injection is completed, the staff starts the shaking device 3, and the shaking device 3 drives the chip storage pool 22 to shake through the driving motor 31, so that the test agent injected into the chip storage unit 2 is fully shaken and the reaction is accelerated; thereby improving the detection efficiency and accuracy of the positive separation circulating tumor cell chip.

[0056] The second reagent storage unit 5 also includes a sample storage structure; the sample storage structure is used to store samples, that is, blood samples to be enriched. The enriched blood samples can be directly dripped on the surface of the positive separation circulating tumor cell chip, and then placed in the chip storage unit 2 to start the enrichment operation; the enriched blood sample can also be placed in the sample storage structure and transferred to the chip storage unit 2 through the mobile rack 53. The mobile rack 53 injects the enriched blood sample into the surface of the positive separation circulating tumor cell chip through the hose 54 above it, and when dripping the enriched blood sample on the surface of the positive separation circulating tumor cell chip, it is necessary to drip the enriched blood sample first, and then inject the test reagent into the chip storage pool 22; the enriched blood sample is placed on the test reagent storage rack 51 on the second reagent storage unit 5 through the test reagent tube, and then interacts with the positive separation circulating tumor cell chip; thereby improving the detection effect of the positive separation circulating tumor cell chip.

[0057] Embodiment 2:

[0058] like Figures 1 to 7 As shown; a triangular block 223 is provided inside the chip storage pool 22, and the triangular block 223 is a right-angled triangular block 223, and two right-angled sides of the triangular block 223 are in contact with the inner wall of the chip storage pool 22, and the inclined surface of the triangular block 223 is inclined toward the direction of the slide slot 221;

[0059] The cover plate 222 is provided with two limiting belts 224 at one end inside the chip storage pool 22, and the two limiting belts 224 are located on both sides of the cover plate 222; the chip storage pool 22 is provided with two winding shafts 225 on the inner wall close to the slide groove 221, and when the cover plate 222 is fully opened, the two winding shafts 225 are respectively located directly below the two limiting belts 224, and the two limiting belts 224 are respectively wound on the corresponding winding shafts 225.

[0060] Here’s how it works:

[0061] On the basis of the above-mentioned embodiment 1, a triangular block 223 is arranged inside the chip storage pool 22, and the triangular block 223 is a right-angled triangular block 223, and the two right-angled sides of the right-angled triangular block 223 are in contact with the bottom wall and the side wall of the chip storage pool 22; so that the inclined surface of the triangular block 223 exists as the bottom wall of the chip storage pool 22, and after the positive separation circulating tumor cell chip is placed inside the chip storage pool 22, the positive separation circulating tumor cell chip is in an inclined state, and the horizontal height of the end of the positive separation circulating tumor cell chip close to the chute 221 is lower than the end away from the chute 221; then the staff injects the test reagent liquid into the chip storage pool 22, and after the injection is completed, the staff manually pushes the cover plate 222, and the cover plate 222 moves inside the chute 221 toward the inside of the chip storage pool 22;

[0062] During the process, two limiting belts 224 are arranged at one end of the cover plate 222 located at the chip storage pool 22, and the two limiting belts 224 are respectively located at both sides of the lower end of the cover plate 222, and two reeling shafts 225 are arranged on the inner wall of the chip storage pool 22. When the cover plate 222 is in a fully opened state, the two reeling shafts 225 are located directly below the corresponding two limiting belts 224, and one end of the limiting belt 224 is wound around the reeling shaft 225; so that when the cover plate 222 moves toward the inside of the chip storage pool 22, the cover plate 222 will pull the limiting belt 224, and the reeling shaft 225 will pull the limiting belt 224. 24 unwinds until the cover plate 222 completely closes the upper end of the chip storage pool 22. At this time, the limiting belt 224 will be in an inclined state, so that the limiting belt 224 can limit the two sides of the positive separation circulating tumor cell chip on the inclined surface of the triangular block 223 to a certain extent, so that when the shaking device 3 shakes the chip storage pool 22, it is prevented from colliding and impacting the positive separation circulating tumor cell chip with the inner wall of the chip storage pool 22, causing the positive separation circulating tumor cell chip to be damaged, thereby improving the detection and use efficiency of the positive separation circulating tumor cell chip;

[0063] When the cover 222 is opened, the cover 222 drives the limiting belt 224 to move toward one end close to the slide slot 221. During the process, the winding shaft 225 winds up the limiting belt 224 until the limiting belt 224 and the side wall of the chip storage pool 22 are parallel to each other; so that the limiting belt 224 no longer limits the positive isolated circulating tumor cell chip, making it convenient for the staff to take out the positive isolated circulating tumor cell chip.

[0064] Embodiment three:

[0065] like Figures 3 to 7 As shown; the two sides of the inclined surface of the triangular block 223 are provided with two mounting grooves 6, the inner part of the mounting groove 6 is slidably connected to the limit plate 61, and a push block 62 is provided below the limit plate 61, and the lower side of the push block 62 is arranged in an arc shape;

[0066] The inner wall of the installation groove 6 is provided with a sliding groove 63, and the push block 62 is slidably connected inside the sliding groove 63;

[0067] The triangular block 223 has two adjustment slots 64 formed inside, the two adjustment slots 64 are respectively located at the lower sides of the two mounting slots 6, and the lower end of the push block 62 is located inside the adjustment slot 64; the inside of the adjustment slot 64 is slidably connected with a slider 65 through a spring, and one end of the slider 65 close to the sliding slot 63 is provided with an inclined surface;

[0068] A rectangular groove 66 is formed at one end of the triangular block 223 away from the slide groove 221, and an arc groove 67 is formed inside the triangular block 223. One end of the arc groove 67 is connected to the rectangular groove 66, and the other end is connected to the adjustment groove 64.

[0069] The arc-shaped groove 67 is slidably connected with an arc-shaped block 68, one end of which is located in the rectangular groove 66, and the other end is located in the adjusting groove 64. A push plate 69 is provided in the rectangular groove 66, and the push plate 69 is connected to the end of the arc-shaped block 68 located in the rectangular groove 66; the end of the arc-shaped block 68 located in the adjusting groove 64 is in contact with the slider 65;

[0070] A rolling ball 621 is disposed at the arc-shaped end surface of the lower end of the pushing block 62 ; when the sliding block 65 moves, the rolling ball 621 contacts the inclined surface on the sliding block 65 .

[0071] Here’s how it works:

[0072] Two mounting grooves 6 are provided on the inclined surface of the triangular block 223, and a limiting plate 61 is slidably connected inside the mounting groove 6. After the positive isolated circulating tumor cell chip is placed inside the chip storage pool 22, the positive isolated circulating tumor cell chip is located on the limiting plates 61 on both sides of the inclined surface of the triangular block 223. A sliding groove 63 is provided inside the triangular block 223, and the sliding groove 63 is perpendicular to the inner wall of the mounting groove 6. An adjusting groove 64 is provided inside the triangular block 223, and the adjusting groove 64 is located below the mounting groove 6. The sliding groove 63 is connected to the adjusting groove 64 and the mounting groove 6, and the pushing block 62 provided at the lower end of the limiting plate 61 is slidably connected inside the sliding groove 63, and the arc-shaped end of the lower end of the pushing block 62 is located inside the adjusting groove 64. A sliding block 65 is slidably connected inside the adjusting groove 64 through a spring, and the end of the sliding block 65 close to the pushing block 62 is provided with an inclined surface.

[0073] A rectangular groove 66 is provided at one end of the triangular block 223 away from the slide groove 221, an arc groove 67 is provided inside the rectangular groove 66, and an arc block 68 is slidably connected inside the arc groove 67. It should be noted that the arc block 68 and the arc groove 67 have the same center, and the other end of the arc groove 67 is connected to the adjustment groove 64. The end of the arc block 68 away from the rectangular groove 66 is located inside the adjustment groove 64 and contacts with the end of the slider 65 away from the push block 62; a push plate 69 is provided at one end of the arc block 68 located inside the rectangular groove 66;

[0074] After the test reagent is injected into the chip storage pool 22, the staff pushes the cover plate 222 into the chip storage pool 22. During the process, the horizontal height of the reel 225 is higher than the lowest end of the triangular block 223. When the cover plate 222 completely closes the chip storage pool 22, the limiting belt 224 is parallel to the inclined surface of the triangular block 223, and there is a gap between the limiting belt 224 and the inclined surface of the triangular block 223, so that the limiting belt 224 cannot contact the positive separation circulating tumor cell chip; the limiting belt 224 will not rub against the positive separation circulating tumor cell chip during the movement, avoiding the problem of wear of the positive separation circulating tumor cell chip;

[0075] However, due to the structural features such as the limit plate 61, the installation groove 6, the adjustment groove 64, the arc block 68, the push plate 69, the arc groove 67, etc., when the cover plate 222 is completely closing the chip storage pool 22, the end of the cover plate 222 close to the push plate 69 will push the push plate 69, and the push plate 69 will push the arc block 68 after being pushed, and the arc block 68 moves into the adjusting groove 64, and the arc block 68 pushes the slider 65 in the adjusting groove 64, and the slider 65 moves toward the end close to the pushing block 62, and the inclined surface on the slider 65 squeezes the arc surface at the lower end of the pushing block 62, and the rolling ball 621 at the lower end of the pushing block 62 moves on the inclined surface of the slider 65, so that the slider 65 The pushing block 62 pushes, and the pushing block 62 moves upward inside the sliding groove 63. The pushing block 62 pushes the limiting plate 61, and the limiting plate 61 moves upward on the inclined surface of the triangular block 223, so that the limiting plate 61 pushes the positive separation circulating tumor cell chip above it, until the pushing plate 69 completely enters the rectangular groove 66, and the limiting plate 61 pushes the positive separation circulating tumor cell chip to contact with the lower end surface of the limiting belt 224, so that the limiting plate 61 and the limiting belt 224 limit the positive separation circulating tumor cell chip; so that the space below and above the positive separation circulating tumor cell chip can be in contact with the test reagent liquid, thereby improving the detection effect of the positive separation circulating tumor cell chip;

[0076] And when the cover plate 222 has not completely closed the chip storage pool 22, the movement of the limiting belt 224 does not contact the positive separation circulating tumor cell chip, and thus will not cause wear to it; when the cover plate 222 completely closes the chip storage pool 22, the limiting plate 61 pushes the positive separation circulating tumor cell chip to make the positive separation circulating tumor cell chip contact with the lower end of the limiting belt 224, thereby limiting the positive separation circulating tumor cell chip and avoiding the problem of wear of the positive separation circulating tumor cell chip during the shaking of the chip storage pool 22.

[0077] Embodiment 4:

[0078] In a preferred embodiment of the device proposed by the present invention, the specific enrichment process is further disclosed as follows:

[0079] Step 1, sample loading: PBS buffer, 4% paraformaldehyde, and 0.2% Triton X-100 are stored in the cleaning solution storage box 413, respectively; FITC-anti-CD45 test reagent (50 μL test reagent diluted in 1 mL PBS), PE-anti-CK test reagent (50 μL test reagent diluted in 1 mL PBS), and 2 μg / mL DAPI test reagent are stored in the test reagent storage racks 514 arranged in sequence; the positive isolated circulating tumor cell chip is stored in the slot 1 of the chip storage unit 2; 4 mL of the patient's peripheral blood is directly dripped on the positive isolated circulating tumor cell chip, and the chip is immersed;

[0080] Step 2: Start the device. After incubating 4 mL of peripheral blood and the chip at room temperature for 45 minutes, the multi-channel peristaltic pump transfers the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42. Then, the single-channel peristaltic pump drives the PBS buffer from the cleaning liquid storage box 41 into the chip storage unit 2. After cleaning, the multi-channel peristaltic pump transfers the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42.

[0081] Step 3: Use a single-channel peristaltic pump to drive 200 μL Triton X-100 from the cleaning solution storage box 41 into the chip storage unit 2, incubate at room temperature for 20 min, and react with the positive isolated circulating tumor cell chip. Use a multi-channel peristaltic pump to transfer the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42. Then, use a single-channel peristaltic pump to drive PBS buffer from the cleaning solution storage box 41 into the chip storage unit 2. After cleaning, use a multi-channel peristaltic pump to transfer the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42.

[0082] Each subsequent step was similar, and the test reagents were 4% paraformaldehyde, 0.2% Triton X-100, and BSA solution;

[0083] Step 2: Start the device, and use a single-channel peristaltic pump to drive 200 μL of 4% paraformaldehyde from the cleaning solution storage box 41 into the chip storage unit 2 to react with the positive separation circulating tumor cell chip, and incubate at room temperature for 10 min;

[0084] Step 3: The single-channel peristaltic pump drives the PBS buffer from the cleaning liquid storage box 41 into the chip storage unit 2 to react with the positive separation circulating tumor cell chip, and then the multi-channel peristaltic pump transfers the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42; this step is repeated 3 times.

[0085] Step 4: Use a single-channel peristaltic pump to drive 200 μL Triton X-100 from the cleaning solution storage box 41 into the chip storage unit 2 to react with the positive isolated circulating tumor cell chip. Then use a multi-channel peristaltic pump to transfer the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42 and incubate at room temperature for 10 min. Repeat step 3 3 times.

[0086] Step 5: Use a single-channel peristaltic pump to drive 200 μL of FITC-anti-CD45 test reagent from the test reagent storage rack 51 into the chip storage unit 2 to react with the positive isolated circulating tumor cell chip. Then, use a multi-channel peristaltic pump to transfer the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42 and incubate at room temperature for 10 min. Repeat step 3 3 times.

[0087] Step 6: Use a single-channel peristaltic pump to drive 200 μL of PE-anti-CK test reagent from the test reagent storage rack 51 into the chip storage unit 2 to react with the positive isolated circulating tumor cell chip. Incubate at room temperature for 30 minutes. Then use a multi-channel peristaltic pump to transfer the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42. Repeat step 3 three times.

[0088] Step 7: The single-channel peristaltic pump drives 200 μL of DAPI test reagent from the test reagent storage rack 51 into the chip storage unit 2 to react with the positive isolated circulating tumor cell chip. The single-channel peristaltic pump then transfers the generated waste liquid from the chip storage unit 2 to the waste liquid storage box 42 and incubates at room temperature for 10 min. Repeat step 3 3 times.

[0089] After enrichment is completed, immunofluorescence imaging can be used to detect the number of CTCs. For example, the chip is taken out from the chip storage unit 2 and placed on the stage of a fluorescence microscope; the capture chip is completely scanned using 405 nm, 488 nm, and 561 nm wavelength excitation light in sequence; the scanned image is fitted and selected (CK+ / CD45- / DAPI+) cells are recorded as CTCs cells, and the number is counted.

Claims

1. A fully automatic circulating tumor cell enrichment device, comprising a device body (1), wherein a cover (11) is provided on the body (1); characterized in that: The machine body (1) is provided with: A chip storage unit (2), the chip storage unit (2) comprising a chip storage box (21); two ends of the chip storage box (21) are rotatably connected to the lower part of the interior of the machine body (1); a plurality of chip storage pools (22) are arranged inside the chip storage box (21); A shaking device (3), wherein the shaking device (3) is arranged below the machine body (1), and the shaking device (3) comprises a driving motor (31), an eccentric wheel (32) is arranged on the driving motor (31), a connecting rod (33) is arranged on the surface of the eccentric wheel (32), and the connecting rod (33) is hinged to the lower end of the chip storage box (21); A first reagent storage unit (4), the first reagent storage unit (4) comprising a plurality of cleaning liquid storage boxes (41) and at least one waste liquid storage box (42); the cleaning liquid storage box (41) is connected to the chip storage tank (22) via a connection pipe (43), and a first driving device is arranged between the cleaning liquid storage box (41) and the chip storage tank (22); the waste liquid storage box (42) is connected to the lower end of the chip storage tank (22) via a connection pipe (43), and a second driving device is arranged between the waste liquid storage box (42) and the chip storage tank (22); A second reagent storage unit (5), the second reagent storage unit (5) comprising a plurality of test reagent storage racks (51) arranged in sequence; The transfer device (52) comprises a movable frame (53), the movable frame (53) is slidably connected to the inside of the machine body (1), and one end of the movable frame (53) away from the bottom wall of the machine body (1) is located directly above the chip storage pool (22); the inside of the movable frame (53) is connected to the second reagent storage unit (5) through a hose (54); a third driving device is arranged between the movable frame (53) and the second reagent storage unit (5); A sensor device, the sensor device is located at the bottom of the cleaning liquid storage box (41) and the end of the corresponding connecting tube (43), and at the top of the waste liquid storage box (42) and the front end of the corresponding connecting tube (43); the sensor device is also located at the bottom of the test reagent storage rack (51) and one end of the hose (54) close to the mobile rack (53); A slide groove (221) is provided at one end of the chip storage pool (22) away from the transfer rack. The slide groove (221) is located on the upper side of the chip storage pool (22). A cover plate (222) is slidably connected inside the slide groove (221). In the initial state, the cover plate (222) closes the upper end of the chip storage pool (22). A triangular block (223) is arranged inside the chip storage pool (22), the triangular block (223) is a right-angled triangular block (223), two right-angled sides of the triangular block (223) are in contact with the inner wall of the chip storage pool (22), and the inclined surface of the triangular block (223) is inclined in the direction of the slide groove (221); Two limiting belts (224) are arranged at one end of the cover plate (222) located inside the chip storage pool (22), and the two limiting belts (224) are located on both sides of the cover plate (222); two winding shafts (225) are arranged on the inner wall of the chip storage pool (22) close to the slide groove (221), and when the cover plate (222) is fully opened, the two winding shafts (225) are respectively located directly below the two limiting belts (224), and the two limiting belts (224) are respectively wound around the corresponding winding shafts (225).

2. The fully automatic circulating tumor cell enrichment device according to claim 1, characterized in that: Two installation grooves (6) are provided on both sides of the inclined surface of the triangular block (223); a limit plate (61) is slidably connected inside the installation groove (6); a push block (62) is provided below the limit plate (61); and the lower side of the push block (62) is arranged in an arc shape; The inner wall of the installation groove (6) is provided with a sliding groove (63), and the pushing block (62) is slidably connected inside the sliding groove (63); The triangular block (223) has two adjustment grooves (64) formed inside, the two adjustment grooves (64) are respectively located at the lower sides of the two mounting grooves (6), and the lower end of the push block (62) is located inside the adjustment groove (64); a slide block (65) is slidably connected inside the adjustment groove (64) via a spring, and one end of the slide block (65) close to the slide groove (63) is provided with an inclined surface; A rectangular groove (66) is formed at one end of the triangular block (223) away from the slide groove (221), and an arc groove (67) is formed inside the triangular block (223), one end of the arc groove (67) is connected to the rectangular groove (66), and the other end is connected to the adjustment groove (64); An arc block (68) is slidably connected inside the arc groove (67), one end of the arc block (68) is located inside the rectangular groove (66), and the other end is located inside the adjustment groove (64). A push plate (69) is provided inside the rectangular groove (66), and the push plate (69) is connected to one end of the arc block (68) located inside the rectangular groove (66); one end of the arc block (68) located inside the adjustment groove (64) contacts the slider (65).

3. The fully automatic circulating tumor cell enrichment device according to claim 2, characterized in that: A rolling ball (621) is arranged at the arc-shaped end surface at the lower end of the pushing block (62); when the sliding block (65) moves, the rolling ball (621) contacts the inclined surface on the sliding block (65).

4. The fully automatic circulating tumor cell enrichment device according to claim 1, characterized in that: The second reagent storage unit (5) includes a refrigeration device for maintaining the temperature in the test reagent storage rack (51) at a constant temperature of 3-5°C.

5. The fully automatic circulating tumor cell enrichment device according to claim 1, characterized in that: The first driving device is a single-channel peristaltic pump, the second driving device is a multi-channel peristaltic pump, and the third driving device is a single-channel peristaltic pump.

6. The fully automatic circulating tumor cell enrichment device according to claim 1, characterized in that: The cleaning liquid storage box (41), the waste liquid storage box (42), and the second reagent storage unit (5) are each independently a warehouse, a tube, a hole, and a groove structure.

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