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

• US 20090054919A2
• Filed: 04/05/2007
• Published: 02/26/2009
• Est. Priority Date: 03/03/2004
• Status: Abandoned Application

• Current Assignee: Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System)
• Original Assignee: Board of Supervisors of Louisiana State University Agricultural & Mechanical College (Louisiana State University System)
• Inventors: Winters-Hilt, Stephen, Pincus, Seth
• Application Number:
  US11/576,723
• Publication Number:
  US 20080097502A1
• Time in Patent Office: Days
• US Class Current: 606/172
• CPC Class Codes: A61B 17/32093 for skin incisions
• CPC Class Codes: A61B 2090/034 abutting on parts of the device itself
• CPC Class Codes: A61B 2090/0814 Preventing re-use
• CPC Class Codes: A61B 5/150022 for capillary blood or interstitial fluid
• CPC Class Codes: A61B 5/150259 Improved gripping, e.g. with high friction pattern or projections on the housing surface or an ergonometric shape
• CPC Class Codes: A61B 5/150465 Specific design of proximal end
• CPC Class Codes: A61B 5/150916 by blocking components, e.g. piston, driving device or fluid passageway
• CPC Class Codes: A61B 5/15111 Semi-automatically triggered, e.g. at the end of the cocking procedure, for instance by biasing the main drive spring or when reaching sufficient contact pressure, the piercing device is automatically triggered without any deliberate action by the user
• CPC Class Codes: A61B 5/15117 comprising biased elements, resilient elements or a spring, e.g. a helical spring, leaf spring, or elastic strap
• CPC Class Codes: A61B 5/15128 comprising 2D- or 3D-shaped elements, e.g. cams, curved guide rails or threads comprising tooth-shaped elements A61B5/15132
• CPC Class Codes: A61B 5/15144 comprising driving means, e.g. a spring, for retracting the piercing unit into the housing
• CPC Class Codes: A61B 5/7267 involving training the classification device
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: G16B 40/00 ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
• CPC Class Codes: G16C 99/00 Subject matter not provided for in other groups of this subclass

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.

Claims

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.