Ultra-sensitive detection of molecules on single molecule arrays

• US 8,846,415 B2
• Filed: 06/19/2012
• Issued: 09/30/2014
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Quanterix Corporation
• Original Assignee: Quanterix Corporation
• Inventors: Duffy, David C., Ferrell, Evan, Randall, Jeffrey D., Rissin, David M., Walt, David R.
• Primary Examiner(s): LAM, ANN Y
• Application Number:
  US13/527,210
• Publication Number:
  US 20140243223A1
• Time in Patent Office: 833 Days
• Field of Search: None
• US Class Current: 436/518
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to systems and methods for detecting analyte molecules or particles in a fluid sample and in some cases, determining a measure of the concentration of the molecules or particles in the fluid sample. Methods of the present invention may comprise immobilizing a plurality of analyte molecules or particles to form a plurality of complexes, releasing at least a portion of some of the plurality of complexes, determining at least a portion of the plurality of complexes released, and determining a measure of the concentration of the analyte molecules or particles in a fluid sample.

Claims

1. A method of quantifying a molecular species, comprising:

providing a plurality of particles, wherein at least some of particles are associated with at least one molecule of the molecular species;

partitioning at least a portion of the plurality of particles across a plurality of reaction vessels so that a statistically significant fraction of the reaction vessels contain no particles associated with a molecule of the molecular species and a statistically significant fraction of reaction vessels contain at least one particle associated with at least one molecule of the molecular species;

detecting the presence or absence of a molecule of the molecular species in at least some of the reaction vessels and determining the percentage of reaction vessels which contain a molecule of the molecular species; and

determining a measure of the quantity of the molecular species based on the percentage of reaction vessels which contain a molecule of the molecular species.

2. The method of claim 1, wherein either one or no molecules of the molecular species to be detected are immobilized with respect to a single particle.

3. The method of claim 1, further comprising an act of determining the concentration of molecular species in the fluid.

4. The method of claim 1, wherein each of the particles comprise a plurality of binding partners for the molecular species.

5. The method of claim 1, wherein the volume of each of the plurality of reaction vessels is between about 10 attoliters and about 100 picoliters.

6. The method of claim 1, wherein the volume of each of the plurality of reaction vessels is between about 1 femtoliter and about 1 picoliter.

7. The method of claim 1, wherein the volume of each of the plurality of reaction vessels is between about 10 femtoliters and about 100 femtoliters.

8. The method of claim 1, wherein the presence or absence of a particle associated with at least one molecule of the molecular species is detected directly.

9. The method of claim 1, wherein the presence or absence of a reaction vessel containing a particle associated with at least one molecule of the molecular species is detected indirectly.

10. The method of claim 9, wherein each of the plurality of reaction vessels is exposed to at least one precursor labeling agent molecule.

11. The method of claim 10, wherein the at least one precursor labeling agent molecule is converted to a labeling agent molecule when contained in a reaction vessel containing a particle associated with at least one molecule of the molecular species.

12. The method of claim 11, wherein the presence or absence of a molecule of the molecular species in a reaction vessels is detected by detecting the presence or absence of a labeling agent molecule in the reaction vessel.

13. The method of claim 1, wherein the molecular species are proteins.

14. The method of claim 1, wherein the molecular species are nucleic acids.

15. The method of claim 1, wherein the molecular species are small molecules.

17. The method of claim 1, wherein the plurality of particles are partitioned such that a statistically significant fraction of the reaction vessels contain no particles associated with a molecule of the molecular species and a statistically significant fraction of reaction vessels contain one particle associated with one molecule of the molecular species.

18. The method of claim 1, further comprising determining the number of the plurality of reaction vessels and/or fraction of the plurality of reaction vessels that contain or do not contain a particle associated with a molecule of the molecular species.

19. The method of claim 18, wherein the number or fraction of the plurality of reaction vessels that contain a particle associated with a molecule of the molecular species is related to the concentration of species in the fluid.

20. The method of claim 18, wherein the number or fraction of the plurality of reaction vessels that contain a particle associated with a molecule of the molecular species is directly proportional to the concentration of molecular species in the fluid.

16. A method of detecting a molecular species in a fluid sample, comprising:

exposing the fluid sample to a plurality of particles, wherein at least some of the particles associate with molecules of the molecular species;

partitioning at least a portion of the plurality of particles across a plurality of reaction vessels, wherein the plurality of particles are partitioned such that a statistically significant fraction of the reaction vessels contain no particles associated with a molecule of the molecular species and a statistically significant fraction of reaction vessels contain at least one particle associated with at least one molecule of the molecular species to be detected; and

determining the presence or absence of a molecule of the molecular species in at least some of the reaction vessels.

21. The method of claim 16, wherein each of the plurality of particles comprises a plurality of binding partners for the molecular species.

22. The method of claim 16, wherein the volume of each of the plurality of reaction vessels is between about 10 attoliters and about 100 picoliters.

23. The method of claim 16, wherein the volume of each of the plurality of reaction vessels is between about 10 femtoliters and about 100 femtoliters.