ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS

• US 20110212462A1
• Filed: 03/24/2010
• Published: 09/01/2011
• Est. Priority Date: 03/01/2010
• Status: Active Grant

• Current Assignee: Quanterix Corporation
• Original Assignee: Quanterix Corporation
• Inventors: Song, Linan, Chang, Lei, Walt, David R., Duffy, David C., Rissin, David M.
• Granted Patent: US 9,678,068 B2
• Time in Patent Office: Days
• US Class Current: 435/7.1
• CPC Class Codes: G01N 33/5306 Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
• 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

Described herein are systems and methods for the detection of and/or determination of a measure of the concentration of analyte molecules or particles in a fluid sample. In some cases, the systems and methods employ techniques to reduce or limit the negative effects associated with non-specific binding events. Certain methods of the present invention involve associating the analyte molecules at least a first type of binding ligand and at least a second type of binding ligand, and spatially segregating the analyte molecules into a plurality of locations on a surface. The presence of an analyte molecule at or in a location may be determined by determining the presence of both the first type of binding ligand and the second type of binding ligand.

Claims

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

providing the analyte molecules or particles immobilized with respect to a binding surface having affinity for at least one type of analyte molecule or particle, the binding surface forming or contained within one of a plurality of locations on a substrate, such that at least some of the plurality of locations contain at least one analyte molecule or particle and a statistically significant fraction of the plurality of locations do not contain any analyte molecules or particles;

exposing the analyte molecules or particles to a first type of binding ligand having an affinity for a first epitope on the analyte molecules or particles and a second type of binding ligand having an affinity for a second epitope on the analyte molecules or particles;

addressing at least a portion of the plurality of the locations and determining the number of said locations containing a first type of binding ligand and a second type of 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 containing both a first type of binding ligand and a second type of binding ligand.

2. The method of claim 1, wherein the exposing step is performed prior to the analyte molecules or particles being immobilized with respect to the binding surface.

3. The method of claim 1, wherein the exposing step is performed after the analyte molecules or particles have been immobilized with respect to the binding surface.

4. The method of claim 3, wherein the binding surface comprises the surface of a bead contained within one of a plurality of locations on a substrate, and wherein the exposing step is performed prior to the bead being contained within one of the plurality of locations on the substrate.

5. The method of claim 3, wherein the binding surface comprises the surface of a bead contained within one of a plurality of locations on a substrate, and wherein the exposing step is performed after the bead is contained within one of the plurality of locations on the substrate.

12. The method of claim 1, wherein the binding surfaces are presented on a plurality of discrete objects.

13. The method of claim 12, wherein the exposing step is conducted prior to containing the plurality of binding surfaces within a plurality of locations.

14. The method of claim 12, wherein the exposing step is conducted following containing the plurality of binding surfaces within a plurality of locations.

15. The method of claim 12, wherein the discrete objects comprise beads.

16. The method of claim 15, wherein the beads are magnetic.

17. The method of claim 1, wherein the first type of binding ligand and the second type of binding ligand are directly detected.

18. The method of claim 1, wherein the first type of binding ligand and the second type of binding ligand are indirectly detected.

19. The method of claim 1, wherein the first type of binding ligand comprises a first type of enzymatic component and the second type of binding ligand comprises a second type of enzymatic component.

20. The method of any claim 1, further comprising exposing the plurality of locations to a plurality of a first type of precursor labeling agent and a second type of precursor labeling agent.

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

providing a fluid sample containing or suspected of containing a plurality of analyte molecules or particles;

exposing the plurality of analyte molecules or particles to a first type of binding ligand having an affinity for a first epitope on the analyte molecules or particles and a second type of binding ligand having an affinity for a second epitope on the analyte molecules or particles;

spatially separating at least a portion of the analyte molecules or particles into a plurality of locations, such that at least some of the plurality of locations contain at least one analyte molecule or particle and a statistically significant fraction of the plurality of locations do not contain any analyte molecules or particles;

addressing at least a portion of the plurality of locations and determining the number of said locations containing both a first type binding ligand and a second type of 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 containing both a first type of binding ligand and a second type of binding ligand.

7. The method of claim 6, wherein the exposing step occurs prior to the spatially separating step.

8. The method of claim 6, wherein the spatially separating steps occurs prior to the exposing step.

9. The method of claim 6, wherein the plurality of location each comprise a binding surface having an affinity for the plurality of analyte molecules or particles.

10. The method of claim 9, further comprising immobilizing the portion of analyte molecules which were spatially separating into the plurality of locations with respect to the binding surface of the locations.

11. The method of claim 6, further comprising immobilizing the plurality of analyte molecules or particles to a plurality of discrete objects such that a statistically significant fraction of the discrete objects are immobilized with respect to only a single analyte molecule or particle and a statistically significant fraction of the discrete objects are free of any analyte molecules or particles, prior to the exposing and spatially separating steps.

21-46. -46. (canceled)