Devices and methods of cell capture and analysis

Abstract

The present invention provides a device for isolating target biomolecules or cells from samples, particularly biological samples. In particular, the device comprises a loading mixture, which contains the biological sample and a first binding entity that specifically binds to the target biomolecule or target cell; and a micro-channel coated with a second binding entity that binds directly or indirectly to the first binding entity. Methods of capturing, detecting, and/or evaluating target biomolecules or target cells (e.g. cancer cells) in biological samples are also disclosed.

1 . A method for detecting a target in a sample comprising
 contacting a sample with a first binding entity to form a pre-loading mixture, wherein the first binding entity specifically binds to a target entity on the surface of the target,   passing the pre-loading mixture through a micro-channel, wherein the surface of the micro-channel is coated with a second binding entity capable of specifically binding, directly or indirectly, to the first binding entity, and   detecting the presence of the target on the surface of the micro-channel.   
     
     
         2 . The method of  claim 1 , wherein the first binding entity is an antibody or antibody cocktail. 
     
     
         3 . The method of  claim 1 , wherein the target is a rare cell in a biological sample. 
     
     
         4 . The method of  claim 3 , wherein the target cell is a cell present in the biological sample in the ratio of 1 out of 10 10  cells, 1 out of 5×10 7 , or 1 out of 10 4  cells. 
     
     
         5 . The method of  claim 3 , wherein the target cell is a cancer cell selected from a breast cancer cell, a prostate cancer cell, a colorectal cancer cell, a lung cancer cell, a pancreatic cancer cell, a ovarian cancer cell, a bladder cancer cell, a endometrial cancer cell, a cervical cancer cell, a liver cancer cell, a renal cancer cell, a thyroid cancer cell, a bone cancer cell, a lymphoma cancer cell, a melanoma cancer cell, and a non-melanoma cancer cell. 
     
     
         6 . The method of  claim 1 , wherein the first binding entity includes an antibody that specifically binds to an epithelial cell surface marker. 
     
     
         7 . The method of  claim 1 , wherein the first binding entity is an epithelial cell adhesion molecule (EpCAM) antibody. 
     
     
         8 . The method of  claim 3 , wherein the target cell is a breast cancer cell and wherein the first binding entity is an antibody that specifically binds to Her2/neu, EpCAM, MUC-1, EGFR, TAG-12, IGF1R, TACSTD2, CD318, CD104, or N-cadherin, or the first binding entity is an antibody cocktail specifically binding a combination thereof. 
     
     
         9 . The method of  claim 3 , wherein the target cell is a melanoma cancer cell and wherein the first binding entity is an antibody that specifically binds to melanocyte differentiation antigens, oncofetal antigens, tumor specific antigens, SEREX antigens or a combination thereof. 
     
     
         10 . The method of  claim 9 , wherein melanocyte differentiation antigens consist of tyrosinase, gp75, gp100, MelanA/MART1 or Trp2. 
     
     
         11 . The method of  claim 9 , wherein the oncofetal antigens consist of MAGE-A1, MAGE-A4, BAGE, GAGE or NY-ESOT. 
     
     
         12 . The method of  claim 9 , wherein the tumor-specific antigens consist of CDK4 and β-catenin. 
     
     
         13 . The method of  claim 3 , wherein the target cell is a prostate cancer cell and wherein the first binding entity is an antibody that specifically binds to EpCAM, MUC-1, EGFR, PSMA, PSA, TACSTD2, PSCA, PCSA, CD318, CD104, or N-cadherin, or the first binding entity is an antibody cocktail specifically binding a combination thereof. 
     
     
         14 . The method of  claim 3 , wherein the target cell is a colorectal cancer cell and the first binding entity is an antibody that specifically binds to EpCAM, CD66c, CD66e, CEA, TACSTD2, CK20, CD104, MUC-1, CD318, or N-cadherin, or the first binding entity is an antibody cocktail specifically binding a combination thereof. 
     
     
         15 . The method of  claim 3 , wherein the target cell is a lung cancer cell and the first binding entity is an antibody that specifically binds to CK18, CK19, TACSTD2, CD318, CD104, CEA, EGFR, or EpCAM, or the first binding entity is an antibody cocktail specifically binding a combination thereof. 
     
     
         16 . The method of  claim 3 , wherein the target cell is a pancreatic cancer cell and the first binding entity is an antibody that specifically binds to MUC-1, CEA, TACSTD2, CD104, CD318, N-cadherin, MUC-1, or EpCAM, or the first binding entity is an antibody cocktail specifically binding a combination thereof. 
     
     
         17 . The method of  claim 3 , wherein the target cell is an ovarian cancer cell and the first binding entity is an antibody that specifically binds to MUC-1, TACSTD2, CEA, CD318, CD104, N-cadherin, or EpCAM, or the first binding entity is an antibody cocktail specifically binding a combination thereof. 
     
     
         18 . The method of  claim 3 , wherein the target cell is an enothelial bladder cancer cell and the first binding entity is an antibody that specifically binds to CD34, CD146, CD62, CD105, CD106, VEGF receptor, or MUC-1, or the first binding entity is an antibody cocktail specifically binding a combination thereof. 
     
     
         19 . The method of  claim 3 , wherein the target cell is an epithelial bladder cancer cell and the first binding entity is an antibody that specifically binds to TACSTD2, EpCAM, CD318, EGFR, 6B5, N-cadherin or folate binding receptor, or the first binding entity is an antibody cocktail that specifically binds a combination thereof. 
     
     
         20 . The method of  claim 3 , wherein the target cell is a cancer stem cell and the first binding entity is an antibody that specifically binds to CD133, CD135, CD117, or CD34, or the first binding entity is an antibody cocktail that specifically binds a combination thereof. 
     
     
         21 . The method of  claim 3 , wherein the target cell is a circulating cancer cell that expresses mesenchymal antigens and the first binding entity is an antibody that specifically binds to FGFR1, FGFR4, EGFR, folate binding receptor, N-cadherin or MSC, or the first binding entity is an antibody that specifically binds a combination thereof. 
     
     
         22 . The method of  claim 3 , wherein the target cell is a circulating cancer cell that expresses angiogenesis surface antigens and the first binding entity is an antibody that specifically binds to a VEGF receptor. 
     
     
         23 . The method of  claim 1 , wherein the first binding entity is a biotinylated-antibody and the second binding entity is avidin. 
     
     
         24 . The method of  claim 1 , further comprising contacting the pre-loading mixture with a third binding entity, wherein the first binding entity is a primary antibody, the third binding entity is a secondary antibody conjugated to a detectable or capturable entity and specifically binds to the first binding entity, and wherein the second binding entity specifically binds to the third binding entity via the detectable or capturable entity. 
     
     
         25 . The method of  claim 1 , further comprises contacting the pre-loading mixture with a third binding entity, wherein the first binding entity is a primary antibody, the third binding entity is a biotinylated secondary antibody that specifically binds to the first binding entity, and wherein the second binding entity is avidin. 
     
     
         26 . The method of  claim 1 , wherein the first binding entity is a mixture of at least first antibody and second antibody, and wherein the first antibody specifically binds to a first epitope of the target entity and the second antibody specifically binds to a second epitope of the target entity. 
     
     
         27 . The method of  claim 26 , wherein the first antibody specifically binds to a stem cell antigen and the second antibody binds to a cancer cell antigen. 
     
     
         28 . The method of  claim 27 , wherein the first antibody specifically binds to CD133, CD135, CD117, CD34 or combinations thereof. 
     
     
         29 . The method of  claim 26 , wherein the first antibody specifically binds to a mesenchymal marker and the second antibody specifically binds to a cancer cell antigen. 
     
     
         30 . The method of  claim 29 , wherein the first antibody specifically binds to FGFR1, FGFR4, MSC or combinations thereof. 
     
     
         31 . The method of  claim 26 , wherein the first antibody specifically binds to an angiogenesis marker and the second antibody specifically binds to a cancer cell antigen. 
     
     
         32 . The method of  claim 31 , wherein the first antibody specifically binds to a VEGF receptor. 
     
     
         33 . The method of  claim 1 , wherein the micro-channel comprises a population of posts distributed on the surface of the micro-channel in random pattern. 
     
     
         34 . The method of  claim 1 , further comprises cross-linking the target bound to the surface of the micro-channel. 
     
     
         35 . The method of  claim 1 , wherein detecting the presence of the target is carried out by detecting the presence of the first binding entity. 
     
     
         36 . A device comprising
 a micro-channel and a loading mixture,   wherein the micro-channel comprises a population of posts distributed on the surface of the micro-channel in random pattern,   wherein the loading mixture comprises a biological sample suspected of containing a target cell and a first binding entity,   wherein the first binding entity specifically binds to a target entity on the target cell, and   wherein the surface of the micro-channel is coated with a second binding entity that specifically binds, directly or indirectly, to the first binding entity.   
     
     
         37 . The device of  claim 36 , wherein the first binding entity comprises a binding entity mixture having at least a first antibody and a second antibody, and wherein the first antibody specifically binds to a first epitope of the target entity and the second antibody specifically binds to a second epitope of the target entity. 
     
     
         38 . The device of  claim 36 , wherein the biological sample is a physiological fluid or tissue selected from the group consisting of blood, plasma, bone marrow, serum, semen, vaginal secretions, urine, saliva, amniotic fluid, cerebral spinal fluid, synovial fluid, lung lavages, fine needle aspirates (FNA) and biopsy tissue sample. 
     
     
         39 . The device of  claim 36 , wherein the loading mixture further comprises a third binding entity conjugated to a detectable entity, wherein the first binding entity is a primary antibody, the third binding entity is a secondary antibody that specifically binds to the first binding entity and the second binding entity specifically binds to the third binding entity. 
     
     
         40 . The device of  claim 36 , wherein the loading mixture further comprises a third binding entity, wherein the first binding entity is a primary antibody, the third binding entity is a biotinylated secondary antibody that specifically binds to the first binding entity and the second binding entity is avidin. 
     
     
         41 . The method of  claim 1 , wherein detecting the presence of the target cell is carried out by detecting the presence of the third binding entity. 
     
     
         42 . A method for capturing a target cell in a biological sample comprising:
 contacting a biological sample with a first binding entity to form a pre-loading mixture, wherein the first binding entity specifically binds to a target entity on the surface of the target cell,   passing the pre-loading mixture through a micro-channel, wherein the surface of the micro-channel is coated with a second binding entity capable of specifically binding to the first binding entity, and   capturing the target cell on the surface of the micro-channel.   
     
     
         43 . The method of  claim 42 , wherein the target cell is a circulating tumor cell (CTC). 
     
     
         44 . The method of  claim 42 , wherein the captured target cells are detected while attached to the surface of the microchannel. 
     
     
         45 . The method of  claim 42 , wherein the target cell is analyzed in situ or in vitro. 
     
     
         46 . The method of  claim 42 , wherein the target cell is evaluated for aneuploidy. 
     
     
         47 . The method of  claim 46 , wherein the target cell is evaluated for aneuploidy at one or more of chromosomes 1, 3, 4, 7, 8, 11, and/or 17. 
     
     
         48 . The method of  claim 46 , wherein aneuploidy is evaluated by FISH. 
     
     
         49 . A method for evaluating isolated circulating cells for malignancy, comprising: evaluating the circulating cells for aneuploidy independent of cytokeratin (CK) expression. 
     
     
         50 . The method of  claim 49 , wherein the method does not involve cytokeratin staining. 
     
     
         51 . The method of  claim 49 , wherein the cells are evaluated for aneuploidy of chromosomes 1, 3, 4, 7, 8, 11, and/or 17. 
     
     
         52 . The method of  claim 51 , wherein the cells are evaluated for monosomy or trisomy 8, 11, and/or 17. 
     
     
         53 . The method of  claim 51 , wherein the cells are evaluated for monosomy 8, 11, and/or 17. 
     
     
         54 . The method of  claim 49 , wherein aneuploidy is evaluated by FISH. 
     
     
         55 . The method of  claim 49 , further comprising, detecting an additional marker of malignancy. 
     
     
         56 . The method of  claim 49 , wherein the additional marker of malignancy is Her2 expression. 
     
     
         57 . The method of  claim 49 , wherein the cells are CK − . 
     
     
         58 . A method for evaluating a sample for a target entity or biomarker, wherein the method comprises
 contacting a sample with a first binding entity to form a pre-loading mixture, wherein the first binding entity specifically binds to a target entity on the surface of the target,   passing the pre-loading mixture through a micro-channel, wherein the surface of the micro-channel is coated with a second binding entity capable of specifically binding, directly or indirectly, to the first binding entity, and   detecting the presence of the target entity or biomarker on the surface of the micro-channel,   
       wherein the presence of the target entity or biomarker is indicative of a condition or disease. 
     
     
         59 . The method of  claim 58 , wherein the first binding entity is an antibody or antibody cocktail. 
     
     
         60 . The method of  claim 58 , wherein the target entity is a rare cell in a biological entity. 
     
     
         61 . The method of  claim 58 , wherein the method does not involve cytokeratin staining. 
     
     
         62 . The method of  claim 58 , wherein the biomarker is aneuploidy of chromosomes 1, 3, 4, 7, 8, 11, 17 and/or 20. 
     
     
         63 . The method of  claim 62 , wherein the biomarker is monosomy or trisomy 8, 11, and/or 17. 
     
     
         64 . The method of  claim 62 , wherein the biomarker is monosomy 8, 11, and/or 17. 
     
     
         65 . The method of  claim 62 , wherein the biomarker is monosomy or tetrasomy 17 or 20. 
     
     
         66 . The method of  claim 58 , wherein aneuploidy is evaluated by FISH. 
     
     
         67 . The method of  claim 58 , further comprising, detecting an additional marker of malignancy. 
     
     
         68 . The method of  claim 58 , wherein the additional marker of malignancy is Her2 expression. 
     
     
         69 . The method of  claim 58 , wherein the target entity is CK − .