Non-invasive cancer detection and analysis by single-molecule imaging

Abstract

Described are chips for detecting a target in a sample including a microfluidic flow chamber comprising one or more flow channels having a capture surface and at least one micromixer. Described are methods of using this chip wherein targets are identified by total internal reflection fluorescence (TIRF).

1 . A chip for detecting of a target in a sample comprising:
 a microfluidic flow chamber comprising one or more flow channels comprising a capture surface and at least one micromixer wherein the capture surface comprises a binding molecule.   
     
     
         2 . The chip of  claim 1  wherein the capture surface is composed of a material selected from the group comprising glass, silicon, PDMS, polystyrene, polycarbonate, polyvinylchloride, polymethyl methacrylate, a cyclic olefin polymer or a combination thereof. 
     
     
         3 . The chip of  claim 1  wherein the binding molecule is a chemical conjugate. 
     
     
         4 . The chip of  claim 3  wherein the chemical conjugate is a silane based compound. 
     
     
         5 . The chip of  claim 4  wherein the silane based compound is a silane comprising one or more moieties selected from the group comprising an amino, a vinyl, an epoxy, an acryloxy, a methacryloxy, a styryl, an isocyanurate, an ureide, a sulfide, an isocyanate, a mercapto group, or a combination thereof. 
     
     
         6 . The chip of  claim 3  wherein the chemical conjugate comprises:
 (a) one or more reactive groups selected from the group comprising succinimidyl valerate, and also including N-hydroxysuccinimide ester, imidoester, epoxide, isothiocyanate, isocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride, fluorobenzene, carbonate, fluorophenyl ester, or a combination thereof; and 
 (b) one or more passivation groups with or without biotin modification selected from the group comprising polyethylene glycol, polyacrylamide, poly(acrylic acid), poly(N-hydroxyethyl acrylamide), poly(2-hydroxyethyl methacrylate), poly(2-methacryloyloxyethyl phosphorylcholine), poly(vinyl alcohol), poly(vinyl pyrrolidone), hydroxyethylcellulose, hydroxypropyl methylcellulose, dextran, hyaluronic acid, or a combination thereof. 
 
     
     
         7 . The chip of  claim 4  wherein the silane based compound is further conjugated to a chemical compound comprising:
 (a) one or more reactive groups selected from the group comprising succinimidyl valerate, and also including N-hydroxysuccinimide ester, imidoester, epoxide, isothiocyanate, isocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride, fluorobenzene, carbonate, fluorophenyl ester, or a combination thereof; and 
 (b) one or more passivation groups with or without biotin modification selected from the group comprising polyethylene glycol, polyacrylamide, poly(acrylic acid), poly(N-hydroxyethyl acrylamide), poly(2-hydroxyethyl methacrylate), poly(2-methacryloyloxyethyl phosphorylcholine), poly(vinyl alcohol), poly(vinyl pyrrolidone), hydroxyethylcellulose, hydroxypropyl methylcellulose, dextran, hyaluronic acid, or a combination thereof. 
 
     
     
         8 . The chip of  claim 3  wherein the chip comprises a capture molecule covalently conjugated to the capture surface by the chemical conjugate. 
     
     
         9 . The chip of  claim 1  wherein the binding molecule contains a first biotin binding complex selected from the group comprising biotin, avidin, NeutrAvidin, streptavidin, or a combination thereof. 
     
     
         10 . The chip of  claim 9  wherein the chip further comprises a capture molecule that comprises a second biotin complex that is bound to the chip by the first biotin binding complex. 
     
     
         11 . The chip of  claim 1  wherein the one or more micromixer is a passive micromixer selected from the group comprising embedded barriers, staggered herringbone grooves, intersecting channels, lamination, serpentine structure, slanted walls, walls with boxes, twisted channels, surface chemistry, zigzag channels or a combination thereof. 
     
     
         12 . The chip of  claim 1  wherein the micromixer is an active micromixer selected from the group comprising acoustic, dielectophoretic, electrohydrodynamic force, electrokinetic instability, electrokinetic time-pulsed, magnetic, magneto-hydrodynamic force, pressure perturbation, thermal, or a combination thereof. 
     
     
         13 . The chip of  claim 1  wherein the micromixer is a combination of a passive micromixer and an active micromixer. 
     
     
         14 . The chip of  claim 1  wherein the microfluidic flow chamber comprises a material selected from the group comprising polydimethylsiloxane (PDMS), silicon, glass, polystyrene, polycarbonate, polyvinylchloride, polymethyl methacrylate, cyclic olefin copolymer, or a combination thereof. 
     
     
         15 . The chip of  claim 1  wherein the one or more flow channels have a width in the range of 0.025 mm to 10 mm, a length in the range of 0.1 mm to 10 mm, and a height in the range of 0.001 mm to 2 mm. 
     
     
         16 . A system for detecting a target in a sample comprising:
 a chip comprising a microfluidic flow chamber comprising one or more flow channels comprising a capture surface and at least one micromixer wherein the capture surface comprises a binding molecule; and   a total internal reflection fluorescence (TIRF) microscopy system wherein the TIRF microcopy system detects the binding molecule on the chip.   
     
     
         17 . The system of  claim 16  further comprising an external pump able to drive continuous, unidirectional, or bidirectional fluid flow through the flow channels in a single pass or by recirculation. 
     
     
         18 . A method of detecting a target in a sample comprising:
 a) providing a chip comprising a microfluidic flow chamber comprising one or more flow channels comprising a capture surface and at least one micromixer wherein the capture surface comprises a binding molecule;   b) placing a capture molecule specific to a target molecule into the one or more flow channels of the chip so it binds to the first binding molecule;   c) placing a sample containing a target molecule recognized by the capture molecule into the one or more flow channels of a chip;   d) placing a detection molecule specific for the target molecule into one or more flow channels of the chip; and   e) detecting the target molecule.   
     
     
         19 . The method of  claim 18  wherein the chip is connected to an external pump able to drive continuous, either unidirectional or bidirectional, fluid flow through the flow channels, either in a single pass or recirculation format. 
     
     
         20 . The method of  claim 18  wherein the target molecule is detected digitally in the sample by total internal reflection fluorescence (TIRF) microscopy. 
     
     
         21 . The method of  claim 18  wherein the detection molecule is labeled with a fluorophore and binds to the target creating a fluorophore-labeled target complex. 
     
     
         22 . The method of  claim 21  wherein the fluorophore-labeled target complex is individually detected. 
     
     
         23 . The method of  claim 20  wherein a total internal reflection fluorescence (TIRF) signal is generated that is collected by an electron multiplying charge coupled device camera with single photon sensitivity. 
     
     
         24 . The method of  claim 18  wherein the capture molecule is conjugated to the chip surface by the first binding molecule having:
 (a) one or more reactive groups selected from the group comprising: succinimidyl valerate, N-hydroxysuccinimide ester, imidoester, epoxide, isothiocyanate, isocyanate, sulfonyl chloride, aldehyde, carbodiimide, acyl azide, anhydride, fluorobenzene, carbonate, fluorophenyl ester, or a combination thereof; and 
 (b) one or more passivation groups with or without biotin modification, selected from the group comprising polyethylene glycol, polyacrylamide, poly(acrylic acid), poly(N-hydroxyethyl acrylamide), poly(2-hydroxyethyl methacrylate), poly(2-methacryloyloxyethyl phosphorylcholine), poly(vinyl alcohol), poly(vinyl pyrrolidone), hydroxyethylcellulose, hydroxypropyl methylcellulose, dextran, hyaluronic acid, or a combination thereof. 
 
     
     
         25 . The method of  claim 18  wherein the first binding molecule is selected from the group comprising biotin, avidin, streptavidin, or NeutrAvidin, or a combination thereof. 
     
     
         26 . The method of  claim 24  wherein the capture molecule comprises a second binding molecule that is able to form a biotin-associated complex selected from the group comprising biotin, avidin, streptavidin, or NeutrAvidin, or a combination thereof. 
     
     
         27 . The method of  claim 18 , wherein the capture molecule is selected from the group comprising an antibody, a peptide, a protein, a nucleic acid, a lipid, a carbohydrate, an aptamer, or a combination. 
     
     
         28 . The method of  claim 18  wherein one or more flow channels is a positive control. 
     
     
         29 . The method of  claim 18  wherein one or more flow channels is a negative control. 
     
     
         30 . The method of  claim 18  wherein the target molecule is a tumor-specific nucleocytoplasmic protein. 
     
     
         31 . The method of  claim 18  wherein the target molecule is a mutant oncoprotein selected from the group comprising RAS, BRAF, PIK3CA, EGFR, NOTCH1 or a combination thereof. 
     
     
         32 . The method of  claim 18  wherein the target molecule is a mutant tumor suppressor protein selected from the group comprising P53, CDKN2A, PTEN, RB, APC, SMAD, ARID1A, MLL2, MLL3, GATA3, VHL, PBRM1 or a combination thereof. 
     
     
         33 . The method of  claim 18  wherein the target molecule is a pathogen-encoded oncoprotein derived from an oncogenic pathogen selected from the group comprising HPV, EBV, HBV, HCV, HTLV-1, KSHV, Merkel cell polyomavirus, or a combination thereof. 
     
     
         34 . The method of  claim 18  wherein the target molecule is P53. 
     
     
         35 . The method of  claim 18  wherein the sample is selected from the group consisting of whole blood, plasma, serum, RBC fraction, urine, saliva, cerebrospinal fluid, semen, sweat, bile, gastric contents, breast milk, exudates, ascites, lymph, sputum, lavage fluid, and bronchial fluid. 
     
     
         36 . The method of  claim 18  wherein the sample is a human sample. 
     
     
         37 . The method of  claim 18  wherein the detection molecule comprises a label selected from the group comprising: a fluorophore, a colorimetric label, a radioactive label, a luminescence label, an electromagnetic label or a combination thereof. 
     
     
         38 . The method of  claim 18 , wherein the first binding molecule is avidin, streptavidin, or NeutraAvidin. 
     
     
         39 . The method of  claim 18 , wherein the detection molecule is selected from the group comprising an antibody, peptide, protein, nucleic acid, lipid, carbohydrate, or aptamer, or a combination thereof. 
     
     
         40 . A method of detecting a composition in a clinical sample comprising:
 a) providing a chip comprising a capture surface comprising a first binding molecule; and a microfluidic flow chamber comprising one or more flow channels comprising the capture surface and at least one micromixer;   b) placing a capture molecule specific to a composition into the one or more flow channels of the chip so it binds to the first binding molecule;   c) placing a sample potentially containing a composition into one or more flow channels of the chip;   d) placing a detection molecule specific for the composition into one or more flow channels of the chip; and   e) detecting the composition in the sample by total internal reflection fluorescence (TIRF) microscopy.   
     
     
         41 . The method of  claim 40  wherein the chip is connected to an external pump able to drive continuous, either unidirectional or bidirectional, fluid flow through the flow channels, either in a single pass or recirculation format. 
     
     
         42 . The method of  claim 40  wherein a TIRF signal is generated by the total internal reflection fluorescence (TIRF) microscopy that is collected by an electron multiplying charge coupled device camera with single photon sensitivity. 
     
     
         43 . The method of  claim 42  wherein the detection molecule is labeled with a fluorophore and binds to the composition creating a fluorophore-labeled biomarker complex. 
     
     
         44 . The method of  claim 42  wherein a fluorophore-labeled composition complex is detected digitally. 
     
     
         45 . The method of  claim 46 , wherein the composition is a biomarker and detecting single biomarker molecules. 
     
     
         46 . The method of  claim 40 , further comprising step f) quantifying the composition by digital image analysis of total internal reflection fluorescence (TIRF) microscopy data. 
     
     
         47 . The method of  claim 18 , further comprising step g) quantifying the target molecule by digital image analysis of total internal reflection fluorescence (TIRF) microscopy data.