Method for enriching and screening a target substance, such as cells, from sample

[0059] In one embodiment, the rotation angle parameter of the multi-channel network matrix 20 is expressed as [0:9:81], which means that the rotation angle of the multi-channel network matrix 20 relative to the upper multi-channel network matrix 20 is 9°. The number of the network-like bases 20 is ten. According to another embodiment, the rotation angle parameter of the multi-channel network matrix 20 is expressed as [0:180], which means that the multi-channel network matrix 20 is rotated 180° relative to the upper multi-channel network matrix 20, and the multi-channel network matrix 20 is rotated by 180°. The number of the shaped bases 20 is two.

[0060] The specific specifications of the mesh cable 23 and the screen hole 22 are mainly described below.

[0061] The shape of the cross section of the screen hole 22 is one or more of square, rectangle, triangle, polygon, circle, parallelogram or trapezoid. In this article, as attached Image 6 As shown, the thickness D of the mesh wire 23 is 10 to 500 μm, preferably 20 to 200 μm, more preferably 20 to 50 μm, and more preferably 40 to 50 μm.

[0062]Here, the thickness of the mesh wire 23 is 10-500 μm, preferably 20-200 μm, more preferably 20-50 μm, and more preferably 40-50 μm. The cross-sectional area of ​​the sieve holes 22 is 10 to 6000 μm 2 , preferably 20 to 3000 μm 2 , more preferably 700 to 3000 μm2, more preferably 700 to 2000 μm 2. The distance between two adjacent layers of porous network matrix 20 is 0.1-10 mm, preferably 0.5-1.5 mm.

[0063] In order to be able to specifically bind to the target 3 , the capture layer includes a capture 21 that can specifically bind to the target 3 . Specifically, the capture objects 21 are bound to the mesh substrate by physical adsorption and/or chemical bonding. Preferably, the capture objects 21 are attached to the mesh substrate by using traut's reagent or thiolate molecules with biotin-avidin. as attached Figure 5 As shown, the capture objects 21 can specifically bind to targets such as cells, bacteria or biomolecules 3 .

[0064] The capture object 21 herein is a specific antibody or other type of specific recognition molecule against target cells, bacteria or target molecules, such as antibodies, bacteriophages, and aptamers, and the target molecules for which the capture object is directed include: EpCAM, EphB4, EGFR, HER2, HER-2/neu, MUC-1, folate receptor, AFP, CEA, Cyfra21-1, TPA, TPS, NMP22, β2-MG, thyroglobulin, ferritin, CA19-9, CA125 , CA50, CA72-4, CA242, CA15-3, SCC, LDH, NSE, PSA, ER, progesterone receptor, HCG, β-hCG, prolactin, ACTH, calcitonin, DHEA-S, cortisol, Aldosterone, uPA/PAI-1, serotonin, 5-oxindoleacetic acid, HGH, FSH, LH, TSH, paraprotein, thymidine kinase, neopterin, SEA, protein S-100, M2-PK, troponin Chromosomal protein A, bone-specific alkaline phosphatase, deoxypyridinoline, CASA, epinephrines, catecholamines, homovanillic acid, epinephrines, vanillylmandelic acid, f-PSA, PCA3, AFP, placental alkaline phosphate Enzymes, calcitonin, gastrin, TSA, AFU, γGT, ALP, CA549, PAP, this-week proteins, and various bacterial and other types of target molecules.

[0065] The captured object 21 is specifically determined according to the target object 3 . The following table lists, without limitation, the targets that can be enriched and screened by the device of the present invention and the corresponding disease types.

[0066]

[0067]

[0068]

[0069] In order to improve the capture efficiency and increase the specificity of the porous network matrix 20, the present invention can also set the traps 21 on the two-layer porous network matrix 20 in the enrichment screening mechanism to be different, or the three-layer porous network matrix The capture objects 21 on the substrate 20 are different. Preferably, the capture objects 21 on each layer of the porous network matrix 20 are different.

[0070] Herein, the capture objects 21 on at least two layers of porous network substrates 20 in the enrichment screening mechanism specifically bind to the same target cells, bacteria or biomolecules 3 respectively; The captured objects 21 are respectively combined with the same target cells, bacteria or biomolecules 3 specifically; more preferably, the captured objects 21 on the four-layer porous mesh substrate 20 are respectively associated with the same target cells, bacteria or biomolecules 3 Specific binding; most preferably, the capture objects 21 on each layer of the porous network substrate 20 are specifically bound to the same target cell, bacteria or biomolecule 3 respectively.

[0071] According to one embodiment, the number of porous mesh substrates 20 is five, and the capture objects 21 on each porous network substrate 20 are different. CEA and ferritin (ferritin) antibodies, these five traps 21 all specifically bind to the same target cell, namely breast cancer tumor cells.

[0072] Herein, in order to screen different kinds of target cells, bacteria or biomolecules 3 at one time, the capture objects 21 of at least one porous network substrate 20 of the plurality of porous network substrates 20 are combined with one target cell, bacteria or The biomolecules 3 are specifically bound, and at least one of the capture objects 21 of the porous network substrate 20 is specifically bound to another target cell, bacteria or biomolecule 3 . Preferably, the capture objects 21 of each porous network matrix 20 specifically bind to different target cells, bacteria or biomolecules 3 respectively.

[0073] According to another embodiment, the number of porous mesh substrates 20 is 10, and the capture objects 21 on each porous network substrate 20 are different, and the target molecules 3 specifically bound by the capture objects 21 of the 10 layers are listed as follows. : HER-2/neu (serum, tissue) - breast cancer; folate receptor (FR) - lung cancer; NMP22 (urine) - bladder cancer; prolactin (PRL) - pituitary tumor; DHEA-S - adrenal cortex; 5- Serotonin (5-HT) - carcinoid; protein S-100 - melanoma; M2-PK - kidney cancer; chromogranin A (CGA) - neuroendocrine tumor; PAP - prostate cancer. The 10 different traps 21 specifically bind to 10 different target molecules respectively or bind to different target cell surfaces (when target molecules are expressed on the outer surface of the target cells).

[0074] The present invention will be further described in detail below with reference to specific embodiments. The raw materials that are not specifically described in the examples are all obtained commercially. Operations that do not specifically mention temperature are carried out at room temperature. For the operation methods and conditions that are not specifically stated, well-known or conventional methods and conditions in the art can be used.

[0075] Example 1

[0076] An enrichment screening mechanism, wherein the angle rotation parameter of the plurality of porous network substrates 20 is [0:36:324], that is, the rotation angle of the lower layer porous network substrate 20 relative to the upper layer porous network substrate 20 is 36°, and there are 10 porous mesh bases 20 in total.

[0077] The distance between adjacent multi-channel mesh substrates 20 is 1 mm, the thickness of the mesh wire 23 is 22 μm, the thickness is 22 μm, the cross section of the sieve hole 22 is square, and the cross-sectional area of ​​the sieve hole 22 is 54×54 μm 2.

[0078] The capture objects on each porous network substrate 20 are EpCAM antibodies, and the method for connecting the capture objects on the porous network substrate 20 includes the following steps:

[0079] (1) Take 4.6 microliters of Traut's reagent solution (0.2 mg/ml), 5 microliters of EPCAM antibody (Cat#ab32392, Abcam), 40.4 microliters of PBS-EDTA (2.5 mmol, pH8.0) and mix the three , incubate for one hour at room temperature;

[0080] (2) dropping the solution incubated in step (1) onto the porous mesh substrate 20, and incubating at room temperature for one hour;

[0081] (3) after adding 1 milliliter of PBS solution to the porous network matrix 20 to rinse, suck off the residual solution;

[0082] (4) repeat step (3);

[0083] (5) 1 ml of 2% BSA solution (2% of BSA solution and 98% of BSA solution in PBS) was added to the porous network matrix 20, and then incubated at room temperature for 30 minutes.

[0084] (6) The porous mesh base 20 is installed and fixed in the cavity of the main body.

[0085] The enrichment screening mechanism prepared above is applied to capture circulating tumor cells, including the following steps:

[0086] (1) Inject 0.5 mL of cell suspension containing 10 lung cancer cells into the enrichment screening mechanism;

[0087] (2) Make 0.5 mL of cell suspension flow through the inlet 11 of the enrichment screening mechanism at a speed of 7.8×10-5m/s, and the cell suspension flows through 10 layers of porous mesh matrix 20 in sequence, so that the lung cancer cells can be combined on enrichment screening mechanisms;

[0088] (3) adding 0.5 ml of PBS solution, and washing the porous network matrix 20 at a speed of 100 microliters per minute to remove unbound sundries, cells or molecules;

[0089] (4) Remove the porous network matrix 20, and place the removed porous network matrix 20 in 0.5 mL of eluent (trypsin accounts for 0.25% of the total mass of the eluent, 1 mM EDTA and the balance of PBS solution) medium, incubate at 37°C for 2 minutes, then shake for 10 seconds to separate lung cancer cells from the enrichment screening mechanism;

[0090] (5) Add 0.5 mL of neutralizing solution (fetal bovine serum), take out the porous mesh matrix 20, centrifuge at 300 g for 5 minutes, remove the supernatant, resuspend the cell pellet with PBS or cell culture medium Counting was performed, and the number of intercepted lung cancer cells was 8.

[0091] Example 2

[0092] It is basically the same as Example 1, except that the angle rotation parameter of the plurality of porous network substrates 20 is [0:9:81], that is, the lower porous network substrate 20 is relative to the upper porous network substrate 20. The angle of rotation is 9°.

[0093] The number of lung cancer cells retained in this example was 5.