Cell-free assay device and methods of use

Abstract

Provided herein is a cell-free assay device, sometimes comprising a lipid bilayer and an endopeptidase assay component, for characterizing a pore forming protein. In some embodiments provided herein is an apparatus comprising a pressure system for characterizing an interaction. Also, provided herein are methods for using a cell-free assay device to characterize a pore forming protein and/or a test substance.

1 - 212 . (canceled) 
     
     
         213 . A device, comprising:
 a substrate comprising a channel and a binding agent in association with the substrate;   a component configured to detect an interaction between an analyte and the binding agent; and   a component configured to apply or maintain a pressure differential across the channel in the device.   
     
     
         214 . The device of  claim 213 , wherein the binding agent is attached to the channel. 
     
     
         215 . The device of  claim 214 , wherein the attachment to the channel comprises a covalent attachment. 
     
     
         216 . The device of  claim 214 , wherein the attachment to the channel comprises a non-covalent attachment. 
     
     
         217 . The device of  claim 214 , wherein the attachment to the channel comprises a linker. 
     
     
         218 . The device of  claim 214 , wherein the binding agent specifically binds to the analyte. 
     
     
         219 . (canceled) 
     
     
         220 . The device of  claim 213 , wherein:
 the device comprises a first volume and a second volume,   the channel separates the first volume and the second volume, and   the pressure differential comprises a difference in pressure between the first volume and the second volume.   
     
     
         221 . The device of  claim 213 , wherein the component configured to detect an interaction is configured to detect a change in current, impedance, resistance, ion current rectification or electrical potential difference. 
     
     
         222 . The device of  claim 213 , wherein the component configured to detect an interaction is configured for a current rectification measurement, an impedance based measurement, a coulter-counter technique or combination thereof. 
     
     
         223 . The device of  claim 213 , wherein the component configured to detect an interaction between the analyte and the binding agents is configured for a current rectification measurement. 
     
     
         224 . The device of  claim 213 , wherein the component configured to apply or maintain a pressure differential is configured to apply a positive pressure across the channel. 
     
     
         225 . The device of  claim 213 , wherein the component configured to apply or maintain a pressure differential is configured to apply a negative pressure across the channel. 
     
     
         226 - 229 . (canceled) 
     
     
         230 . The device of  claim 213 , wherein the substrate and the channel comprises a coating. 
     
     
         231 . (canceled) 
     
     
         232 . The device of  claim 213 , wherein the analyte is chosen from an antibody, antibody fragment, reduced antibody, chemically modified antibody, antibody receptor, antigen, hapten, anti-hapten, protein, cleaved protein, polypeptide, peptide, nucleic acid, a nucleotide, a nucleotide analog or derivative, an alkyl moiety, an alkanoyl moiety, an alkanoic acid or alkanoate moiety, a glyceryl moiety, a phosphoryl moiety, a glycosyl moiety, an ubiquitin moiety, lectin, aptamer, receptor, ligand, metal ion, a pathogen, bacterium, anthrax, virus, biomarker, chemical contaminant, organic contaminant, drug, toxin, ricin, chemical compound, or combinations thereof. 
     
     
         233 . The device of  claim 232 , wherein the analyte is nucleic acid. 
     
     
         234 . The device of  claim 232 , wherein the analyte is an antigen. 
     
     
         235 . The device of  claim 232 , wherein the analyte is a protein. 
     
     
         236 . The device of  claim 232 , wherein the analyte is a cleaved protein. 
     
     
         237 . The device of  claim 213 , wherein the binding agent is chosen from an antibody, antibody fragment, reduced antibody, chemically modified antibody, antibody receptor, antigen, hapten, anti-hapten, protein, cleaved protein, polypeptide, peptide, nucleic acid, a nucleotide, a nucleotide analog or derivative, an alkyl moiety, an alkanoyl moiety, an alkanoic acid or alkanoate moiety, a glyceryl moiety, a phosphoryl moiety, a glycosyl moiety, an ubiquitin moiety, lectin, aptamer, receptor, ligand, metal ion, a pathogen, bacterium, anthrax, virus, biomarker, chemical contaminant, organic contaminant, drug, toxin, ricin, chemical compound or combinations thereof. 
     
     
         238 . The device of  claim 237 , wherein the binding agent is an antibody. 
     
     
         239 . The device of  claim 237 , wherein the binding agent is an aptamer. 
     
     
         240 . The device of  claim 237 , wherein the binding agent is a protein. 
     
     
         241 . The device of  claim 237 , wherein the binding agent is a nucleic acid. 
     
     
         242 . The device of  claim 213 , wherein the component configured to detect an interaction is configured for a fluorescence loss, gain, polarization, or FRET based measurement, or combination thereof. 
     
     
         243 . The device of  claim 232 , wherein the analyte is a biomarker. 
     
     
         244 . The device of  claim 232 , wherein the analyte is a drug. 
     
     
         245 . A method for altering an interaction between an analyte and a binding agent, comprising interacting an analyte with a binding agent in association with a substrate in a system, where the substrate comprises a channel, inducing a pressure differential across the channel and assessing an interaction between the analyte and the binding agent, whereby inducing a pressure differential alters the interaction relative to the interaction assessed without inducing a pressure differential. 
     
     
         246 . The method of  claim 245 , wherein altering the interaction between and analyte and a binding agent comprises altering the rate of interaction between an analyte and a binding agent, whereby inducing a pressure differential alters the rate of interaction relative to the rate of interaction assessed without inducing a pressure differential.