Method of coupling binding agents to a substrate surface

Abstract

The present invention relates to a method of coupling multiple binding agents to respective areas of a substrate surface by hydrodynamic addressing, using two laminar fluid flows that flow together in the same direction over the substrate surface with an interface to each other to successively couple the binding agents to the substrate areas, wherein each successive coupling of a binding agent to a surface area is followed or preceded by selective deactivation or activation of a selected surface area according to a defined protocol. The invention also relates to the use of such a binding agent-coupled substrate surface for analytical purposes.

1 . A method of coupling at least two different binding agents to respective defined areas of a substrate surface by hydrodynamic addressing based on two laminar fluid flows that flow together in the same direction over the substrate surface with an interface to each other, and selectively contacting a defined area of the substrate surface with a desired fluid by positioning the interface laterally through adjustment of the relative flow rates of the two fluid flows, which method comprises at least one of hydrodynamic addressing procedures A and B, wherein: 
 procedure A comprises immobilizing a first binding agent to a first area of the substrate surface by contacting the area with a fluid containing the first binding agent, deactivating the first area by subjecting the area to a deactivating fluid, and immobilizing a second binding agent to a second area of the substrate surface by contacting a substrate surface area including the first and second areas with the second binding agent; and    procedure B comprises deactivating a first area of the substrate surface by subjecting the area to a deactivating fluid, immobilizing a first binding agent to a second area of the substrate surface by contacting the first and second areas with a fluid containing the first binding agent, activating at least a part of the first area by subjecting the area to an activating fluid, and immobilizing a second binding agent to the first area by contacting the area with a fluid containing the second binding agent.    
     
     
         2 . The method according to  claim 1 , wherein procedure A comprises the steps of: 
 a) providing a substrate surface, at least part of which is reactive to permit coupling of binding agents thereto;    b) passing over the substrate surface a laminar flow of a fluid containing a first binding agent, and adjacent thereto a laminar flow of a blocking fluid that does not interact with the substrate surface, such that the two fluids flow together in the same direction with an interface to each other, and adjusting the relative flow rates of the two laminar fluid flows to position the interface such that the first binding agent-containing fluid selectively contacts a first reactive area of the substrate surface to couple the first binding agent thereto;    c) replacing the flow of the first binding agent-containing fluid with a laminar flow of a deactivating fluid, and positioning the interface laterally such that the deactivating fluid selectively contacts at least the first binding agent-coupled area but less than the whole reactive surface area for deactivation thereof; and    d) replacing the flow of the deactivating fluid with a laminar flow of a fluid containing the second binding agent, and positioning the interface laterally such that the second binding agent-containing fluid selectively contacts the deactivated area and an adjacent second reactive area of the substrate surface to selectively couple the second binding agent to the second area.    
     
     
         3 . The method according to  claim 2 , which additionally comprises the step of: 
 e) replacing the flow of the second binding agent-containing fluid with a laminar flow of deactivating fluid, and positioning the interface laterally such that the deactivating fluid selectively contacts at least the deactivated area and the second binding agent-coupled area of the substrate surface for deactivation thereof.    
     
     
         4 . The method according to  claim 3 , wherein steps b) to e) are repeated at least once to couple at least one additional binding agent to a respective reactive area on the substrate surface.  
     
     
         5 . The method according to  claim 1 , wherein procedure B comprises the steps of: 
 a) providing a substrate surface, at least part of which is reactive to permit coupling of binding agents thereto;    b) passing over the substrate surface a laminar flow of a deactivating fluid, and adjacent thereto a laminar flow of a blocking fluid that does not interact with the substrate surface, such that the two fluids flow together in the same direction with an interface to each other, and adjusting the relative flow rates of the two laminar fluid flows to position the interface such that the deactivating fluid selectively contacts a first reactive area of the substrate surface for deactivation thereof;    c) replacing the flow of the deactivating fluid with a laminar flow of a fluid containing a first binding agent, and positioning the interface laterally such that the first binding agent-containing fluid selectively contacts the deactivated area and an adjacent second reactive area of the substrate surface to selectively couple the first binding agent to the second area;    d) replacing the flow of the first binding agent-containing fluid with a laminar flow of activating fluid, and positioning the interface laterally such that the activating fluid selectively contacts at least a part of the deactivated first area for activation thereof; and    e) replacing the flow of the activating fluid with a laminar flow of a fluid containing a second binding agent, and positioning the interface laterally such that the second binding agent-containing fluid selectively contacts the activated first area to selectively couple the second binding agent thereto.    
     
     
         6 . The method according to  claim 5 , wherein steps b) to e) are repeated at least once to couple at least one additional binding agent to a respective reactive area on the substrate surface.  
     
     
         7 . The method according to  claim 1 , wherein at least one of the reactive areas of the substrate surface comprises activated functional groups.  
     
     
         8 . The method according to  claim 2 , wherein step a) comprises passing a laminar flow of activating fluid over at least a part of the substrate surface to provide the at least partly reactive surface.  
     
     
         9 . The method according to  claim 5 , wherein step a) comprises passing a laminar flow of activating fluid over at least a part of the substrate surface to provide the at least partly reactive surface.  
     
     
         10 . The method according to  claim 2 , wherein step a) comprises providing a surface with pre-activated functional groups.  
     
     
         11 . The method according to  claim 5 , wherein step a) comprises providing a surface with pre-activated functional groups.  
     
     
         12 . The method according to  claim 1 , wherein at least one of the reactive areas of the substrate surface comprises functional groups capable of reacting with the binding agents without activation of the functional groups.  
     
     
         13 . The method according to  claim 2 , which comprises the step of, prior to performing step b), passing over the substrate surface a laminar flow of deactivating fluid and an adjacent laminar flow of blocking fluid, and positioning the interface such that the deactivating fluid selectively contacts a reactive edge area of the substrate surface adjacent to the first area to be contacted with binding agent in step b) for deactivation thereof.  
     
     
         14 . The method according to  claim 3 , which comprises performing steps b) to e) in the opposite lateral direction to the flow path to couple at least one additional binding agent to a respective reactive area of the substrate surface.  
     
     
         15 . The method according to  claim 14 , which comprises activating an additional part of the substrate surface prior to performing steps b) to e) of  claim 2  in the opposite lateral direction to the flow path.  
     
     
         16 . The method according to  claim 3 , wherein in steps c) and e) the laminar flow of deactivating fluid is adjusted to contact also a reactive area adjacent to the binding agent-coupled area to provide a non-coupled area between neighbouring binding agent-coupled areas.  
     
     
         17 . The method according to  claim 1 , wherein procedure B comprises immobilizing an additional binding agent to a third area situated on the other side of the second area immobilized with the first binding agent by selectively contacting the third area with a fluid containing the additional binding agent.  
     
     
         18 . The method according to  claim 17 , wherein the third area is activated prior to immobilizing the additional binding agent.  
     
     
         19 . The method according to  claim 1 , which comprises the steps of: 
 a) passing over the substrate surface a laminar flow of an activating fluid, and adjacent thereto a laminar flow of a blocking fluid that does not interact with the substrate surface, and positioning the interface between the two fluids such that the activating fluid selectively contacts a part of the substrate surface for activation thereof;    b) replacing the flow of activating fluid with a laminar flow of blocking fluid, replacing the blocking fluid with a laminar flow of a fluid containing a first binding agent, and positioning the interface such that the fluid containing the first binding agent selectively contacts the non-activated part of the surface and an adjacent first area of the activated part of the surface to couple the first binding agent to the first area;    c) replacing the flow of the first binding agent-containing fluid with a laminar flow of blocking fluid, replacing the blocking fluid with a laminar flow of a fluid containing a second binding agent, and positioning the interface such that the binding agent-containing fluid selectively contacts a second area of the activated part of the surface adjacent to the first area coupled with the first binding agent to couple the second binding agent to the second area;    d) replacing the flow of the second binding agent-containing fluid with a laminar flow of a deactivating fluid, and positioning the interface such that the deactivating fluid selectively contacts the first and second areas coupled with the first and second binding agents, respectively;    e) replacing the flow of deactivating fluid with a laminar flow of blocking fluid, replacing the flow of blocking fluid with a laminar flow of activating fluid, and positioning the interface such that the activating fluid selectively contacts at least a portion of the non-activated part of the surface, adjacent to the part activated in step a), for activation thereof; and    f) replacing the flow of blocking fluid with a laminar flow of a fluid containing a third binding agent, replacing the flow of activating fluid with a laminar flow of blocking fluid, and positioning the interface such that the binding agent-containing fluid selectively contacts the first and second areas coupled with the first and second binding agents, respectively, and a third area comprising at least a part of the surface portion activated in step e) to couple the third binding agent to the third area.    
     
     
         20 . The method according to  claim 19 , which additionally comprises the step of: 
 g) replacing the flow of binding agent-containing fluid with a laminar flow of a deactivating fluid, and positioning the interface such that the deactivating fluid contacts at least the area coupled with the third binding agent for deactivation thereof.    
     
     
         21 . The method according to  claim 19 , which comprises coupling a fourth binding agent to a remaining activated area adjacent to the third area after coupling of the third binding agent.  
     
     
         22 . The method according to  claim 1 , which comprises the steps of: 
 a) passing over the substrate surface a laminar flow of a fluid containing an activating fluid, and adjacent thereto a laminar flow of a blocking fluid that does not interact with the substrate surface, and positioning the interface between the two fluids such that the activating fluid selectively contacts a part of the substrate surface for activation thereof;    b) replacing the flow of activating fluid with a laminar flow of blocking fluid, replacing the blocking fluid with a laminar flow of a fluid containing a first binding agent, and positioning the interface such that the fluid containing the first binding agent selectively contacts the non-activated part of the surface and an adjacent first area of the activated part of the surface to couple the first binding agent to the first area;    c) replacing the flow of the first binding agent-containing fluid with a laminar flow of blocking fluid, replacing the blocking fluid with a laminar flow of a fluid containing a second binding agent, and positioning the interface such that the binding agent-containing fluid selectively contacts a second area of the activated part of the surface adjacent to the first area coupled with the first binding agent to couple the second binding agent to the second area;    d) replacing the flow of binding agent-containing fluid with a laminar flow of blocking fluid, replacing the flow of blocking fluid with a laminar flow of activating fluid, and positioning the interface such that the activating fluid selectively contacts at least a portion of the non-activated part of the surface, adjacent to the part activated in step a), for activation thereof; and    e) replacing the flow of activating fluid with a laminar flow of a fluid containing a third binding agent, and positioning the interface such that the binding agent-containing fluid selectively contacts the area activated in step d) to couple the third binding agent thereto.    
     
     
         23 . The method according to  claim 22 , which additionally comprises the step of: 
 f) replacing the flow of binding agent-containing fluid with deactivating fluid, and positioning the interface such that the deactivating fluid contacts at least areas coupled with the binding agents and previously not deactivated for deactivation thereof.    
     
     
         24 . The method according to  claim 22 , which comprises coupling a fourth binding agent to a remaining activated area adjacent to the third area after coupling of the third binding agent.  
     
     
         25 . The method according to  claim 22 , which comprises deactivating a part of the surface portion activated in step d), and after coupling the third binding agent in step e), reactivating the deactivated area and coupling a fourth binding agent thereto.  
     
     
         26 . The method according to  claim 1 , wherein the substrate surface is a sensing surface of a sensor.  
     
     
         27 . The method according to  claim 26 , wherein the sensing surface is provided in a flow cell.  
     
     
         28 . The method according to  claim 27 , wherein the flow cell comprises two inlets and one outlet.  
     
     
         29 . A method of analysing a fluid sample for at least one analyte, comprising sensitising a sensing surface with at least two different binding agents according to the method of  claim 1 , contacting the sensitised areas with the fluid sample, and detecting interaction between the analyte and the sensing surface.  
     
     
         30 . A method of analysis, comprising sensitising a sensing surface with at least two different binding agents according to the method of  claim 1 , contacting the sensitised areas with at least one analyte, and studying interaction between the analyte and the sensing surface.  
     
     
         31 . The method of  claim 1 , wherein a system comprising: a flow cell having an inlet end and an outlet end, at least one sensing surface on a wall surface within the flow cell located between the inlet and outlet ends, wherein the flow cell has at least two inlet openings at the inlet end and at least one outlet opening at the outlet end, such that separate laminar flows entering the flow cell through the respective inlet openings can flow side by side in the same direction through the flow cell over the sensing surface, means for applying laminar flows through the inlet openings, such that the laminar flows pass side by side through the flow cell over the sensing surface with an interface to each other that is parallel to the direction of the laminar flows; and means for varying the relative flow rates of the laminar fluids to displace laterally the interface over the sensing surface; is used for for performing the method.  
     
     
         32 . A computer program product comprising program code means for performing the steps of  claim 1  when the program is run on a computer.  
     
     
         33 . A computer program product stored on a computer readable medium, comprising a readable program for causing processing means in or associated with a system comprising: a flow cell having an inlet end and an outlet end, at least one sensing surface on a wall surface within the flow cell located between the inlet and outlet ends, wherein the flow cell has at least two inlet openings at the inlet end and at least one outlet opening at the outlet end, such that separate laminar flows entering the flow cell through the respective inlet openings can flow side by side in the same direction through the flow cell over the sensing surface, means for applying laminar flows through the inlet openings, such that the laminar flows pass side by side through the flow cell over the sensing surface with an interface to each other that is parallel to the direction of the laminar flows; and means for varying the relative flow rates of the laminar fluids to displace laterally the interface over the sensing surface; to control the execution of the steps  claim 1.