Method and system for detecting a target within a population of molecules

Patent metadata

Field	Value
Publication number	US-11275083-B2
Application number	US-201916240839-A
Country	US
Kind code	B2
Filing date	Jan 7, 2019
Priority date	Oct 27, 2006
Publication date	Mar 15, 2022
Grant date	Mar 15, 2022

Abstract

A method of detecting a target within a population of molecules comprising: contacting a plurality of labeled probe molecules with the population of molecules potentially containing a target of the probe molecules; acquiring a probe specific signal emitted by said labeled probe molecules that bound to said target together with a background signal; preferentially modulating said probe specific signal by at least one of modulating said acquisition and modulating an emission of said probe specific signal; and detecting said probe specific signal over said background signal using said preferential modulation.

First claim

What is claimed is:

1. A method of detecting at least one target molecule within a population of molecules, the method comprising:
contacting a plurality of probe molecules, selected to bind specifically with the target molecule, each probe molecule labeled with at least one fluorescent molecule, with the population of molecules containing at least one of said target molecule, where at least one probe molecule binds to at least one target molecule;
contacting the plurality of probe molecules with magnetic beads, wherein the at least one probe molecule that binds to the target molecule, or a part of that probe molecule, also binds to one of the magnetic beads via an intermediary component;
forming a cloud of the magnetic beads, condensed;
illuminating the cloud of magnetic beads with an excitation beam that produces fluorescent emission from the fluorescent molecules labeling the probe molecules or parts of probe molecules bound to the magnetic beads; acquiring a background signal and a signal of the fluorescent emission;
detecting the fluorescent emission signal over the background signal; and
determining from the detected fluorescent emission signal, one or both of a presence of the target molecule within the population of molecules, and an amount of the target molecules within the population of molecules;
wherein the method is an energy transfer-based assay, wherein at least one of the probe molecules includes a probe molecule labeled with at least one or more fluorescent molecules at a first end thereof and having one or more quenching molecules at a second end thereof; and
wherein, prior to detecting the fluorescent signal, the one or more fluorescent molecules are separated from the one or more quenching molecules.

2. The method according to claim 1 , wherein said contacting a plurality of probe molecules with the population of molecules potentially containing at least one of said target molecule includes at least one probe molecule selectively binding to at least one target molecule.

3. The method according to claim 1 , wherein said contacting the plurality of probe molecules with magnetic beads includes at least one magnetic bead selectively binding with a probe molecule via at least one intermediary component.

4. The method according to claim 1 , wherein the intermediary component is one or more of the following: biotin, a DNA sequence, an RNA sequence, an antibody, a protein, and a target molecule.

5. The method according to claim 1 , wherein the at least one probe molecule binds with the target molecule before the magnetic bead binds with the probe molecule or part of the probe molecule via an intermediary component.

6. The method according to claim 1 , wherein the at least one probe molecule binds with the target molecule after the magnetic bead binds with the probe molecule or part of the probe molecule via an intermediary component.

7. The method of claim 1 , wherein the one or more fluorescent molecules are separated from the one or more quencher molecules by PCR (polymerase chain reaction).

8. The method according to claim 1 , wherein the method is a sandwich-based assay, wherein a capture probe, labeled with a portion of the intermediary component, and a signaling probe, labeled with a fluorescent molecule, hybridize to the target molecule.

9. The method of claim 1 , wherein the fluorescent signal is detected by a CCD camera.

10. The method according to claim 1 , wherein said at least one target molecule includes at least one molecule type selected from the group consisting of a nucleic acid sequence, an amino acid sequence, a carbohydrate, a carbohydrate sequence, an ion, and a feature of a protein determined by non-primary structure.

11. The method according to claim 1 , wherein at least some of said plurality of probe molecules include at least one of nucleic acids, peptides, proteins, antibodies, and ion chelators; and
wherein at least some of said plurality of target molecules include at least one molecule type selected from the group consisting of a nucleic acid sequence, an amino acid sequence, a carbohydrate, a carbohydrate sequence, an ion, and a feature of a protein determined by non-primary structure.

12. The method according to claim 1 , wherein said at least one probe molecule bound to said at least one target molecule includes at least one protein molecule bound to one protein target.

13. The method according to claim 1 , wherein said at least one probe molecule bound to said at least one target molecule includes at least one nucleic acid molecule bound to a nucleic acid target.

14. The method according to claim 1 , wherein said at least one probe molecule bound to said at least one target molecule includes at least one antibody bound to an epitope.

15. The method according to claim 1 , wherein the intermediary component through which the probe molecule or part of the probe molecule binds to one of the magnetic beads does not comprise any part of the target molecule.

16. The method according to claim 1 wherein at least a first portion of said plurality of probe molecules each have a fluorescent label and are bound to a target and wherein at least a second portion of said probe molecules are each attached to a magnetic bead and are bound to the same target.

17. The method according to claim 16 , wherein the probe molecules bound to the target molecules include at least one protein molecule bound to one protein target.

18. The method according to claim 16 , wherein the probe molecules bound to the target molecules include at least one nucleic acid molecule bound to a nucleic acid target.

Assignees

• Univ Ramot

Inventors

• Arie Ady
• Danielli Amos

Classifications

• G01N21/6428 Primary

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N33/54353

  with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393) · CPC title

• G01N33/54333 Primary

  Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction · CPC title

• C12Q1/6818

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• G01N21/1717

  with a modulation of one or more physical properties of the sample during the optical investigation, e.g. electro-reflectance · CPC title