Real-time analytical methods and systems

• US 9,746,476 B2
• Filed: 06/10/2015
• Issued: 08/29/2017
• Est. Priority Date: 06/12/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Korlach, Jonas, Turner, Stephen, Flusberg, Benjamin, Chaisson, Mark, Schadt, Eric, Wegener, Jeffrey
• Primary Examiner(s): Gonzalez, Antonio Galisteo
• Application Number:
  US14/735,408
• Publication Number:
  US 20150362503A1
• Time in Patent Office: 811 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 2458/10 Oligonucleotides as tagging agents for labelling antibodies
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6875 Nucleoproteins

Abstract

The present invention is generally directed to compositions, methods, and systems for performing single-molecule, real-time analysis of a variety of different biological reactions, and for determining various characteristics of the different biological reactions. The ability to analyze such reactions provides an opportunity to study those reactions as well as to potentially identify factors and/or approaches for impacting such reactions, e.g., to stimulate, enhance, or inhibit such reactions.

Claims

1. A method of monitoring passage of a reaction component across a portion of an amphipathic aggregate, the method comprising:

a) immobilizing a single amphipathic aggregate at a reaction site, wherein at least one molecule is imbedded in the single amphipathic aggregate and wherein the single amphipathic aggregate comprises an aggregate-confined reaction volume localized within a micelle, within a liposome, or within a structural confinement covered by a lipid bilayer;

b) exposing the single amphipathic aggregate to a reaction mixture comprising a labeled reaction component, wherein the labeled reaction component binds the imbedded molecule, wherein the binding results in the labeled molecule being transported into the aggregate-confined reaction volume, and wherein the labeled reaction component comprises a detectable label that emits a signal when present in the aggregate-confined reaction volume; and

c) detecting the signal at the reaction site, wherein the signal at the reaction site is indicative that the labeled reaction component has passed into the aggregate-confined reaction volume, thereby monitoring the passage of the labeled reaction component across a portion of the amphipathic aggregate.

2. The method of claim 1, wherein the single amphipathic aggregate is a single lipid bilayer within a nanohole.

3. The method of claim 1, wherein the reaction site is within a nanohole.

4. The method of claim 1, wherein the single amphipathic aggregate is immobilized at the reaction site through association with one or more binding agents at the reaction site.

5. The method of claim 1, wherein the reaction mixture comprises multiple labeled reaction components.

6. The method of claim 1, wherein the reaction site is on a substrate comprising an array of reaction sites.

7. The method of claim 1, wherein the label is fluorescent and the method further comprises:

providing excitation illumination capable of exciting the fluorescent label at the reaction site; and

detecting emission from the fluorescent label.

8. The method of claim 1, wherein the single amphipathic aggregate comprises multiple imbedded molecules.

9. The method of claim 8, wherein the multiple imbedded molecules comprise a plurality of a single type of imbedded molecule.

10. The method of claim 8, wherein the multiple imbedded molecules comprise more than one type of imbedded molecule.

11. The method of claim 1, wherein the reaction mixture comprises multiple labeled molecules capable of interacting with the imbedded molecule.

12. The method of claim 11, wherein the multiple labeled molecules comprise a plurality of a single type of labeled molecule.

13. The method of claim 11, wherein the multiple labeled molecules comprise more than one type of labeled molecule, wherein each type of labeled molecule is differentially labeled to be distinguishable from every other type.

14. The method of claim 1, wherein the reaction site is on a substrate comprising an array of reaction sites.

15. The method of claim 14, wherein a plurality of different single amphipathic aggregates having different imbedded molecules are immobilized to the array of reaction sites, wherein single reaction sites in the array of reaction sites contain only a single amphipathic aggregate.