Arrays Of Biological Membranes And Methods And Use Thereof

Abstract

The present invention overcomes the problems and disadvantages associated with prior art arrays by providing an array comprising a plurality of biological membrane microspots associated with a surface of a substrate that can be produced, used and stored, not in an aqueous environment, but in an environment exposed to air under ambient or controlled humidities. Preferably, the biological membrane microspots comprise a membrane bound protein. Most preferably, the membrane bound protein is a G-protein coupled receptor, an ion channel, a receptor serine/threonine kinase or a receptor tyrosine kinase.

1 . An array comprising a plurality of biological membrane microspots associated with a surface of a substrate. 
     
     
         2 . The array of  claim 1 , wherein the biological membrane microspots comprise a membrane bound protein. 
     
     
         3 . The array of  claim 2 , wherein the membrane bound protein is a G-protein coupled receptor (GPCR). 
     
     
         4 . The array of  claim 3 , wherein the biological membrane microspots comprise different GPCRs, said GPCRs being member of a GPCR subfamily, having similar tissue distribution or similar physiological or pharmacological function. 
     
     
         5 . The array of  claim 2 , wherein the membrane bound protein is an ion channel. 
     
     
         6 . The array of  claim 3 , wherein the GPCR comprises an intracellular domain and the GPCR is oriented such that the intracellular domain faces the surface of the substrate. 
     
     
         7 . The array of  claim 3 , wherein the GPCR comprises an extracellular domain and the GPCR is oriented such that the extracellular domain faces the surface of the substrate. 
     
     
         8 . The array of  claim 2 , wherein the membrane bound protein is a receptor tyrosine kinase. 
     
     
         9 . The array of  claim 2 , wherein the membrane bound protein is a receptor serine/threonine kinase. 
     
     
         10 . The array of  claim 2 , wherein the membrane bound protein is a receptor guanylate cyclase. 
     
     
         11 . The array of  claim 1 , wherein the substrate comprises glass, metal, silicon, or plastic. 
     
     
         12 . The array of  claim 1 , wherein the substrate is configured as a chip, a slide or a microplate. 
     
     
         13 . The array of  claim 1 , wherein the surface is coated. 
     
     
         14 . The array of  claim 13 , wherein the coating is a material that enhances the affinity of the biological membrane microspot or the membrane bound protein for the substrate. 
     
     
         15 . The array of  claim 14 , wherein the material confers a contact angle ranging from about 15° to 80°. 
     
     
         16 . The array of  claim 14 , wherein the material is a silane, thiol, disulfide, or a polymer. 
     
     
         17 . The array of  claim 14 , wherein the thiol is on a substrate comprising a gold-coated surface. 
     
     
         18 . The array of  claim 16 , wherein the thiol comprises hydrophobic and hydrophilic moieties. 
     
     
         19 . The array of  claim 18 , wherein the thiol is a thioalkyl compound. 
     
     
         20 . The array of  claim 16 , wherein the silane is on a substrate comprising glass. 
     
     
         21 . The array of  claim 16 , wherein the silane presents terminal polar moieties. 
     
     
         22 . The array of  claim 21 , wherein the terminal polar moieties are hydroxyl, carboxyl, phosphate, sulfonate, or amino groups. 
     
     
         23 . The array of  claim 21 , wherein the surface is positively charged and contains amino groups. 
     
     
         24 . The array of  claim 14 , wherein the material is γ-aminopropylsilane. 
     
     
         25 . The array of  claim 14 , wherein the material is a lectin. 
     
     
         26 . The array of  claim 25 , wherein the surface is coated with wheat germ agglutinin. 
     
     
         27 . The array of  claim 14 , wherein the material is an antibody having specific binding affinity for a membrane protein. 
     
     
         28 . The array of  claim 27  wherein the material is an antibody having specific binding affinity for a G protein. 
     
     
         29 . The array of  claim 14 , wherein the material is a derivatized monolayer. 
     
     
         30 . The array of  claim 29 , wherein the derivatized monolayer comprises covalently bonded linker moieties. 
     
     
         31 . The array of  claim 29 , wherein the monolayer is a self-assembled monolayer (SAM). 
     
     
         32 . The array of  claim 29 , wherein the monolayer comprises a thioalkyl compound or a silane compound. 
     
     
         33 . The array of  claim 31 , wherein the thioalkyl is selected from the group consisting of a thioalkyl acid, thioalkyl alcohol, thioalkyl amine, and halogen containing thioalkyl compound. 
     
     
         34 . The array of  claim 32 , wherein the compound is a thioalkyl acid. 
     
     
         35 . The array of  claim 33 , wherein the thioalkyl compound is 16-mercaptohexadecanoic acid. 
     
     
         36 . The array of  claim 34 , wherein the silane compound is selected from the group consisting of a silyl anhydride, silyl acid, silyl amine, silyl alcohol, silyl thiol, vinyl silane or silyl acrylate. 
     
     
         37 . The array of  claim 29 , wherein the linker moiety comprises a straight or branched C 10 -C 25  alkyl, alkynyl, alkenyl, aryl, araalkyl, heteroalkyl, heteroalkynyl, heteroalkenyl, heteroaryl, heteroaraalkyl molecule comprising:
 (i) a terminal functional group capable of reacting with the derivatized monolayer;   (ii) a hydrophilic spacer region; and   (iii) a hydrophobic membrane adhering region.   
     
     
         38 . The array of  claim 37 , wherein the terminal functional group is selected from the group consisting of a carboxylic acid, halogen, amine, thiol, alkene, acrylate, anhydride, ester, acid halide, isocyanate, hydrazine, maleimide and hydroxyl group. 
     
     
         39 . The array of  claim 37 , wherein the hydrophilic spacer region comprises n oxyethylene groups, wherein n=2 to 25. 
     
     
         40 . The array of  claim 37 , wherein the membrane adhering region comprises a straight or branched chain C 10 -C 25  hydrophobic tail. 
     
     
         41 . The array of  claim 31 , wherein the SAM is an alkanethiol modified with a silane. 
     
     
         42 . The array of  claim 41 , wherein the alkanethiol is selected from the group consisting of 11-mercaptoundecanol, 11-mercaptoundecanoic acid, 11-mercaptoundecylamine, 16-mercaptohexadecanol, 16-mercaptohexadecanoic acid. 
     
     
         43 . The array of  claim 41 , wherein the silane is 3-aminopropyltriethoxysilane. 
     
     
         44 . The array of  claim 41 , wherein the substrate is gold. 
     
     
         45 . The array of  claim 13 , wherein the coating comprises nitrilotriacetic acid or ethylenediamine triacetic acid groups and the biological membrane microspots comprise GPCRs having a histidine tag at the c-terminus. 
     
     
         46 . The array of  claim 1 , wherein the surface is porous. 
     
     
         47 . The array of  claim 1 , wherein the substrate is selected from the group consisting of glass, silicon, polymeric materials, and metallic substrates. 
     
     
         48 . A method for producing an array comprising:
 providing a substrate having a surface;   providing a solution a biological membrane;   immersing the tip of a pin into the solution;   removing the tip from the solution to provide a solution adhered to the tip;   contacting the solution with the surface to thereby transfer the solution from the tip to the surface; and   repeating the contacting step a plurality of times to provide biological membrane microspots patterned in an array on the surface.   
     
     
         49 . The method of  claim 48 , wherein the solution comprises a protein. 
     
     
         50 . The method of  claim 49 , wherein the protein is a G-protein coupled receptor. 
     
     
         51 . The method of  claim 49 , wherein the protein is an ion channel. 
     
     
         52 . The method of  claim 48 , further comprising the step of contacting the microspot with a solution comprising a protein. 
     
     
         53 . The method of  claim 48 , wherein the surface of the substrate is exposed to air under ambient or controlled humidities when the tip of the pin contacts the substrate. 
     
     
         54 . A method for detecting a binding event between a probe and target compound, said method comprising:
 contacting a solution comprising the target compound with an array of probe biological membrane microspots associated with a surface of a substrate, the target compound having one or more constituents, and detecting a binding event between at least one or more of the probes with one or more of the constituents of the target.   
     
     
         55 . The method of  claim 54 , wherein at least one of the constituents of the target is labeled and the detection step comprises detecting the presence of the label. 
     
     
         56 . The method of  claim 55 , wherein the detection of the label is carried out by imaging based on the radioactivity, fluorescence, phosphorescence, chemiluminescence, or resonance light scattering emanating from the bound target. 
     
     
         57 . The method of  claim 55 , further comprising washing the substrate of unbound target prior to the detection step. 
     
     
         58 . The method of  claim 54 , wherein the array of microspots is incubated with labeled cognate target and an unlabeled target compound, and the binding event between the unlabeled target compound and the probe is determined by measuring a decrease in the signal of the label due to competition between the cognate labeled target and the unlabeled target compound for the probe. 
     
     
         59 . The method of  claim 58 , wherein the labeled cognate target is incubated with the array before incubation with the unlabeled target. 
     
     
         60 . The method of  claim 58 , wherein the target is unlabeled and the binding event is determined by a change in physical properties at the interface. 
     
     
         61 . The method of  claim 60 , wherein the change in physical properties at the interface is a change in refractive index or electrical impedence. 
     
     
         62 . The method of  claim 54 , wherein the target is unlabeled and the binding of the target is detected by mass spectroscopy. 
     
     
         63 . The method of  claim 54 , wherein the probe biological membrane microspots comprise a G-protein coupled receptor. 
     
     
         64 . The method of  claim 54 , wherein the probe biological membrane microspots comprise a ion channel. 
     
     
         65 . The method of  claim 63 , wherein the target is unlabeled and the binding of the target is detected by measuring levels of labeled nonhydrolyzable GTP. 
     
     
         66 . The method of  claim 65 , wherein the nonhydrolyzable GTP is GTPγS. 
     
     
         67 . The method of  claim 66 , wherein the GTPγS is labeled with a radioactive isotope or a fluorescent compound. 
     
     
         68 . The method of  claim 67 , wherein the GTPγS is [ 35 s] GTPγS or BODIPY-FL-GTPγS. 
     
     
         69 . The method of  claim 54 , wherein the probe biological membrane microspots comprise a receptor tyrosine kinase. 
     
     
         70 . An array comprising a plurality of biological membrane microspots associated with a surface of a glass substrate, wherein the surface is coated with γ-aminopropylsilane and the biological membrane microspots comprise a G-protein coupled receptor. 
     
     
         71 . An array comprising a plurality of biological membrane microspots associated with a surface of a substrate coated with an amine terminated compound. 
     
     
         72 . The array of  claim 71 , wherein the substrate comprises glass, metal or plastic. 
     
     
         73 . The array of  claim 71 , wherein the amine terminated compound is a silane. 
     
     
         74 . The array of  claim 73 , wherein the silane is gamma-aminopropyl-silane. 
     
     
         75 . The array of  claim 71 , wherein the amine terminated compound is 11-mercaptoundecylamine and the substrate comprises gold. 
     
     
         76 . The array of  claim 71 , wherein the biological membrane microspots comprise a membrane bound protein. 
     
     
         77 . The array of  claim 76 , wherein the membrane bound protein is a G protein coupled receptor, a receptor tyrosine kinase, an ion channel, a receptor serine/threonine kinase, or a receptor guanylate cyclase. 
     
     
         78 . The array of  claim 71 , wherein the substrate is configured as a chip, a slide or a microplate. 
     
     
         79 . An immobilized membrane comprising a biological membrane associated with a surface of a substrate coated with an amine terminated compound. 
     
     
         80 . The immobilized membrane of claim of  79 , wherein the substrate is glass, metal or plastic. 
     
     
         81 . The immobilized membrane of claim of  78 , wherein the amine terminated compound is a silane. 
     
     
         82 . The immobilized membrane of claim of  81 , wherein the silane is gamma-aminopropyl-silane. 
     
     
         83 . The immobilized membrane of claim of  79 , wherein the amine terminated molecule is 11-mercaptoundecylamine and the substrate comprises gold. 
     
     
         84 . The immobilized membrane of claim of  79 , wherein the biological membrane comprises a membrane bound protein. 
     
     
         85 . The immobilized membrane of claim of  84 , wherein the membrane bound protein is a G protein coupled receptor, a receptor tyrosine kinase, an ion channel, a receptor serine/threonine kinase, or a receptor guanylate cyclase.