Membrane Based Assays

Abstract

Membrane-based assays using surface detector array devices suitable for use with a biosensor are disclosed. The device is formed of a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions. The bilayer-compatible surface regions carry on them, separated by an aqueous film, supported fluid bilayers. The bilayers may contain selected receptors or biomolecules. A bulk aqueous phase covers the bilayers on the substrate surface. Arrays may be engineered to display natural membrane materials in a native fluid bilayer configuration, permitting high-throughput discovery of drugs that target and affect membrane components. The membrane-based assays detect binding events by monitoring binding-induced changes in one or more physical properties of fluid bilayers.

1 . A method for assaying an interaction between a test agent and a lipid bilayer-associated component, comprising:
 providing a surface detector array device comprising
 (i) a substrate having a surface defining a plurality of distinct bilayer-compatible surface regions separated by one or more bilayer barrier regions, said bilayer-compatible surface regions and said bilayer barrier regions being formed of different materials, and 
 (ii) a plurality of lipid bilayer expanses localized above said plurality of distinct bilayer-compatible surface regions, wherein said lipid bilayer expanses are localized above said surface regions in the absence of covalent linkages between said lipid bilayer expanses and said bilayer-compatible surface regions, and are separated therefrom by an aqueous film interposed between said bilayer-compatible surface regions and said corresponding lipid bilayer expanses, the lipid bilayer expanses having a component associated with the lipid bilayer expanse; 
   contacting said device with a bulk aqueous phase comprising the test agent that specifically binds to the lipid bilayer-associated component, whereby the membrane fluidity of at least one of the plurality of lipid bilayer expanses changes when said test agent binds to said lipid bilayer-associated component;   evaluating the membrane fluidity of one or more of said lipid bilayer expanses, and   detecting binding of the test agent to the lipid bilayer-associated component by correlating a change in membrane fluidity to binding.   
     
     
         2 . The method of  claim 1 , wherein the lipid bilayer-associated component is selected from a protein, a nucleic acid, a glycolipid, a lipopolysaccharide, a sterol, a lipid-linked molecule, and a fatty acid. 
     
     
         3 . The method of  claim 1 , wherein the lipid bilayer-associated component is a bacterial endotoxin. 
     
     
         4 . The method of  claim 1 , further comprising a label attached to one or more of the lipid bilayer expanses. 
     
     
         5 . The method of  claim 4 , wherein said label is attached to a target membrane component. 
     
     
         6 . The method of  claim 4 , wherein said label is attached to a background membrane component. 
     
     
         7 . The method of  claim 4 , wherein said label is selected from the group consisting of a fluorophore, an electron spin resonance label, a radioactive label, a semiconductor nanoparticle label, and a metallic nanoparticle label. 
     
     
         8 . The method of  claim 1 , wherein evaluating the membrane fluidity comprises a method selected from the group consisting of fluorescence recovery after photobleaching, fluorescence anisotropy, fluorescence correlation spectroscopy, fluorescence resonance energy transfer, fluorescence resonance energy transfer microscopy, electrophoresis, and electrical molecular force microscopy. 
     
     
         9 . The method of  claim 1 , wherein the test agent is an antibody. 
     
     
         10 . The method of  claim 1 , wherein the test agent is a small molecule. 
     
     
         11 . The method of  claim 1 , wherein the test agent is a protein. 
     
     
         12 . The method of  claim 1 , wherein the test agent comprises a surface of a cell, a vesicle, a phantom cell, a liposome, a giant vesicle, a lipid-covered glass bead, or a component of any thereof. 
     
     
         13 . The method of  claim 1 , wherein the bulk aqueous phase further comprises a second test agent and further comprising determining whether said second test agent affects the interaction of the test agent with the lipid bilayer-associated component. 
     
     
         14 . A method for assaying an interaction between a test agent and a lipid bilayer-associated component, comprising:
 providing a lipid bilayer expanse comprising a lipid bilayer-associated component; contacting said lipid bilayer expanse with a bulk aqueous phase comprising the test agent that binds to said lipid bilayer-associated component; and   evaluating the membrane fluidity of said lipid bilayer expanse, wherein the membrane fluidity is affected when said test agent binds to the lipid bilayer-associated component, and   detecting binding of the test agent to the lipid bilayer-associated component by correlating a change in membrane fluidity to binding.   
     
     
         15 . The method of  claim 14 , wherein said lipid bilayer-associated component is selected from the group consisting of a protein, a nucleic acid, a glycolipid, a lipopolysaccharide, a sterol, a lipid-linked molecule and a fatty acid. 
     
     
         16 . The method of  claim 14 , wherein said lipid bilayer-associated component is a bacterial endotoxin. 
     
     
         17 . The method of  claim 14 , further comprising a label attached to the lipid bilayer expanses. 
     
     
         18 . The method of  claim 17 , wherein said label is attached to a target membrane component. 
     
     
         19 . The method of  claim 18 , wherein said label is attached to a background membrane component. 
     
     
         20 . The method of  claim 17 , wherein said label is selected from the group consisting of a fluorophore, an electron spin resonance label, a radioactive label, a semiconductor nanoparticle label, and a metallic nanoparticle label. 
     
     
         21 . The method of  claim 14 , wherein said membrane fluidity is evaluated using a method selected from the group consisting of fluorescence recovery after photobleaching, fluorescence anisotropy, fluorescence correlation spectroscopy, fluorescence resonance energy transfer, fluorescence resonance energy transfer microscopy, electrophoresis, and electrical molecular force microscopy. 
     
     
         22 . The method of  claim 14 , wherein the test agent is a small molecule. 
     
     
         23 . The method of  claim 14 , wherein the test agent is a protein. 
     
     
         24 . The method of  claim 14 , wherein the test agent comprises a surface of a cell, a vesicle, a phantom cell, a liposome, a giant vesicle, a lipid-covered glass bead, or a component of any thereof. 
     
     
         25 . The method of  claim 14 , wherein the bulk aqueous phase further comprises a second test agent, and further comprising determining whether said second test agent affects the interaction of the test agent with the lipid bilayer-associated component. 
     
     
         26 . The method of  claim 14 , wherein the test agent is an antibody.