Channel Current Cheminformatics And Bioengineering Methods For Immunological Screening, Single-Molecule Analysis, And Single-Molecular-Interaction Analysis

Abstract

Analytical tools and methods employing processing of channel current blockade measurements to detect or assess changes in environmental conditions generally, and more specifically, to detect and assess interactions between a blockade-producing auxiliary molecule in the channel and either the surrounding conductive medium generally or molecules in that conductive medium. The auxiliary molecule is disposed within the channel in order to generate a highly structured channel current blockade signal. The blockade signal resulting from inducing ionic current flow through the channel with auxiliary molecule present, is based on an auxiliary molecule chosen to be highly susceptible to modulation when the auxiliary molecule interacts with other molecules in the surrounding conductive medium, or when it otherwise is modified or affected at the molecular level by changes in the physical or chemical conditions of that surrounding medium. The tools and methods enable, amongst other things, candidate antibody screening, molecular affinity and bond strength analysis and detection of a variety of changes in test media, even at the molecular level.

1 . A method comprising 
 receiving a blockade channel current signal carried by an ionic current flowing through a partially-blockaded nano-scale membrane channel defined by a membrane which partitions a conductive medium,    exposing the channel to a test condition during ionic current flow while extracting from the blockade channel current signal, a set of one or more pattern features to establish over a period of time either a blockade channel current signal pattern or a change in the blockade channel current signal pattern.    
     
     
         2 . A method according to  claim 2 , further comprising 
 comparing the blockade channel current signal pattern, or the change in the blockade channel current signal pattern, to at least one blockade channel current signal pattern, or at least one change in the blockade channel current signal pattern, associated with at least one known condition, to thereby correlate the test condition with the known condition.    
     
     
         3 . A method according to  claim 2 , wherein the known condition is selected from the group consisting of (i) the presence of a known molecule, (ii) a known molecular characteristic, and (iii) a known environmental condition.  
     
     
         4 . A method according to  claim 3 , wherein the nano-scale membrane channel is partially blockaded by the presence of an auxiliary molecule in the membrane channel.  
     
     
         5 . A method according to  claim 4 , wherein the test condition is comprised of the introduction of a test molecule to the conductive medium.  
     
     
         6 . A method according to  claim 5 , wherein the auxiliary molecule is at least a portion of a single antibody, wherein the test molecule is an antigen, and wherein the known condition is one or more antigen-antibody binding events.  
     
     
         7 . A method according to  claim 6 , wherein the known condition is a plurality of discrete antigen-antibody binding events, and wherein the process further comprises 
 measuring the average length of time during which the one or more discrete antigen-antibody binding events takes place during the period of time.    
     
     
         8 . A method according to  claim 5 , wherein (a) further comprising 
 correlating the blockade channel current signal pattern or the change in the blockade channel current signal pattern with a characterization of interaction between the test molecule and the auxiliary molecule.    
     
     
         9 . A method according to  claim 5 , wherein the auxiliary molecule is an aptamer, wherein the test molecule is a biomolecule, and wherein the known condition is binding affinity.  
     
     
         10 . A method for screening candidate agents for preventing pore formation, comprising 
 in a conductive medium, contacting a membrane with one of the candidate agents and then placing a membrane-permeabilizing agent in the presence of the membrane and the candidate agent under conditions which would permit pore formation in the membrane in the absence of the candidate agent, and    introducing an ionic charge to the membrane in the conductive medium and measuring the time which passes from the moment the membrane-permeabilizing agent is placed in the presence of the membrane until pore formation in the membrane permits an ionic current to flow through the pore to be detected.    
     
     
         11 . A method for identifying an effective cytosolic antigen delivery mechanism for use in evoking a cytotoxic T Lymphocyte response in an organism challenged by one or more cytosolic virulence factors, comprising 
 introducing a single pore-forming toxin to a membrane so as to form at least one pore through which an ionic current may flow, and    causing the current to flow through the pore to generate one or more signals indicative of at least one characteristic of antigen transmembrane transport.    
     
     
         12 . Apparatus comprising 
 a membrane which partitions a conductive medium, the membrane defining at least one nano-scale channel through which an ionic current may flow, the channel having disposed therein an auxiliary molecule which causes modulations in the ionic current when the ionic current flows through the channel,    an ionic current source,    a sensor for measuring the ionic current flowing through the channel and generating a blockade channel current signal,    a computer system programmed and configured for receiving and processing the blockade channel current signal from the sensor to enable the extraction of a set of one or more pattern features.    
     
     
         13 . Apparatus according to  claim 12 , wherein the computer system is programmed and configured to further enable recognition of a pattern of the blockade channel current signal, or a change in the pattern of the blockade channel current signal, over time.  
     
     
         14 . Apparatus according to  claim 13 , wherein the membrane is a solid-state membrane.  
     
     
         15 . Apparatus according to  claim 13 , wherein the membrane is a lipid bi-layer.  
     
     
         16 . Apparatus according to  claim 13  wherein the auxiliary molecule is covalently bound to a first study molecule to produce a first study molecule/auxiliary molecule blockade channel current signal carried by the ionic current flowing through the channel, and the computer system is programmed and configured to detect a change in the pattern of the first study molecule/auxiliary molecule blockade channel current signal upon interaction of the first study molecule with a second study molecule over time, and to correlate the change in the pattern with at least one previously measured pattern, or at least one previously measured change in pattern, associated with a known molecule or a known molecular characteristic, respectively.