ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS

• US 20180017552A1
• Filed: 04/14/2017
• Published: 01/18/2018
• Est. Priority Date: 03/01/2010
• Status: Active Grant

• Current Assignee: Quanterix Corporation
• Original Assignee: Quanterix Corporation
• Inventors: Duffy, David C., Rissin, David M., Walt, David R., Fournier, David, Kan, Cheuk
• Granted Patent: US 10,725,032 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54393 Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

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 with respect to a plurality of capture objects. At least a portion of the plurality of capture objects may be spatially separated into a plurality of locations. A measure of the concentration of analyte molecules in a fluid sample may be determined, at least in part, on the number of reaction vessels comprising an analyte molecule immobilized with respect to a capture object. In some cases, the assay may additionally comprise steps including binding ligands, precursor labeling agents, and/or enzymatic components.

Claims

1. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:

exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or suspected of containing the at least one type of analyte molecules or particles;

immobilizing analyte molecules or particles with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one analyte molecule or particle and a statistically significant fraction of the capture objects do not associate with any analyte molecule or particle;

spatially segregating at least a portion of the capture objects subjected to the immobilizing step into a plurality of separate locations;

addressing at least a portion of the plurality of locations subjected to the spatially segregating step and determining the number of said locations containing an analyte molecule or particle; and

determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of locations determined to contain an analyte molecule or particle.

3. The method of claim 1, wherein the percentage of capture objects which associate with at least one analyte molecule and/or binding ligand is less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, or less than about 5%, or less than about 1%, or less than about 0.5%, or less than about 0.1% or the total number of capture objects.

4. The method of claim 1, wherein the percentage of capture objects which do not associated with any analyte molecules and/or binding ligands is at least about 20%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 95%, or at least about 99%, or at least about 99.5%, or at least about 99.9%, of the total number of capture objects.

5. The method of claim 1, wherein in the addressing step, the number of said locations containing a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle not containing an analyte molecule or particle or a binding ligand is determined.

6. The method of claim 5, wherein the measure of the concentration of analyte molecule or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing an analyte molecule or particle or a binding ligand, to the total number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle.

7. The method of claim 5, wherein the measure of the concentration of analyte molecule or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing an analyte molecule or particle or a binding ligand, to the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle but not to contain any capture objects that include a binding surface having affinity for at least one type of analyte molecule or particle containing an analyte molecule or particle or a binding ligand.

8. The method of claim 1, wherein the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on the ratio of the number of locations addressed in the addressing step determined to contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle containing an analyte molecule or particle or binding ligand, to the number of locations addressed in the addressing step that do not contain a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle.

9. The method of claim 1, wherein the plurality of capture objects that include a binding surface having affinity for at least one type of analyte molecule or particle comprises a plurality of beads.

10. The method of claim 1, wherein the plurality of locations comprises a plurality of reaction vessels.

11. The method of claim 10, wherein the plurality of reaction vessels is formed on the end of a fiber optic bundle.

12. The method of claim 1, wherein at least a portion of the analyte molecules or particles are associated with at least one binding ligand.

13. The method of claim 1, wherein the analyte molecules or particles comprise an enzymatic component.

15. The method of claim 1, wherein the concentration of analyte molecules or particles in the fluid sample is less than about 50×

10<sup>−

15</sup> M, or less than about 40×

10<sup>−

15</sup> M, or less than about 30×

10<sup>−

15</sup> M, or less than about 20×

10<sup>−

15</sup> M, or less than about 10×

10<sup>−

15</sup> M, or less than about 5×

10<sup>−

15</sup> M, or less than about 1×

10<sup>−

15</sup> M.

16. The method of claim 1, wherein the measure of the concentration of analyte molecules or particles in the fluid sample is determined at least in part by comparison of a measured parameter to a calibration standard.

17. The method of claim 1, wherein during the immobilizing step, at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 95% of the analyte molecules or particles are immobilized with respect to a capture object that includes a binding surface having affinity for at least one type of analyte molecule or particle.

19. The method of claim 1, wherein during the spatially segregating step, at least about 5%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the capture objects subjected to the immobilizing steps are spatially separated into the plurality of locations.

20. The method of claim 10, wherein the number of reaction vessels addressed in the addressing step is at least about 5%, or at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the total number of reaction vessels.

2. A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:

exposing a plurality of capture objects that each include a binding surface having affinity for at least one type of analyte molecule or particle, to a solution containing or suspected of containing the at least one type of analyte molecules or particles to form capture objects comprising at least one immobilized analyte molecule or particle;

mixing the capture objects prepared in the exposing step to a plurality of binding ligands such that at least some of the capture objects associate with a single binding ligand and a statistically significant fraction of the capture objects do not associate with any binding ligand;

spatially segregating at least a portion of the capture objects subjected to the mixing step into a plurality of locations;

addressing at least a portion of the plurality of locations subjected to the spatially segregating step and determining the number of locations containing a binding ligand; and

determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of locations determined to contain a binding ligand.

14. The method of claim 2, wherein the binding ligand comprises an enzymatic component.

18. The method of claim 2, wherein during the mixing step, at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% of the immobilized analyte molecules or particles associate with a binding ligand.

21-141. -141. (canceled)