ULTRA-SENSITIVE DETECTION OF MOLECULES ON SINGLE MOLECULE ARRAYS

• US 20100075407A1
• Filed: 09/23/2008
• Published: 03/25/2010
• Est. Priority Date: 09/23/2008
• Status: Active Grant

• Current Assignee: Quanterix Corporation
• Original Assignee: Quanterix Corporation
• Inventors: Ferrell, Evan, Randall, Jeff, Walt, David R., Duffy, David C., Rissin, David M.
• Granted Patent: US 8,222,047 B2
• Time in Patent Office: Days
• US Class Current: 435/287.200
• 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 detecting analyte molecules or particles in a fluid sample, comprising:

exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component and at least one analyte molecule or particle;

dissociating at least a portion of each complex to form a plurality of dissociated species, which are not immobilized with respect to the substrate;

partitioning the plurality of dissociated species across a plurality of reaction vessels; and

determining the presence or absence of a dissociated species in at least one reaction vessel.

2. The method of claim 1, wherein the plurality of dissociated species are partitioned such that a statistically significant fraction of the reaction vessels contain no dissociated species and a statistically significant fraction of reaction vessels contain at least one dissociated species.

3. The method of claim 1, wherein the plurality of dissociated species are partitioned such that a statistically significant fraction of the reaction vessels contain no dissociated species and a statistically significant fraction of reaction vessels contain one dissociated species.

4. The method of claim 2, 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 dissociated species

5. The method of claim 4, wherein the number or fraction of the plurality of reaction vessels that contain a dissociated species is related to the concentration of analyte molecules or particles in the sample.

6. The method of claim 4, wherein the number or fraction of the plurality of reaction vessels that contain a dissociated species is directly proportional to the concentration of analyte molecules or particles in the sample.

7. The method of claim 4, wherein the number or fraction of the plurality of reaction vessels that contain a dissociated species is less than the number of analyte molecules or particles contained in a volume of the sample applied to the substrate in the exposing act.

8. The method of claim 4, further comprising an act of determining the concentration of analyte molecules or particles in the fluid sample.

9. The method of claim 1, wherein the substrate comprises a plurality of beads.

10. The method of claim 9, wherein the beads are magnetic.

11. The method of claim 1, wherein the substrate comprises a microtiter plate.

12. The method of claim 1, wherein the plurality of reaction vessels is formed on the end of a fiber optic bundle.

13. The method of claim 1, wherein the plurality of reaction vessels are formed upon the mating of at least a portion of a sealing component and at least a portion of a second substrate.

14-63. -63. (canceled)

64. A system for detecting analyte molecules or particles in a fluid sample, comprising:

a first substrate comprising a plurality of capture components able to become specifically immobilized with respect to a plurality of analyte molecules or particles of a sample, such that the analyte molecules or particles associate with capture components to form a plurality of complexes, wherein the complexes are susceptible to being dissociated from the first substrate by a dissociating agent to form a plurality of dissociated species; and

a second substrate comprising an array comprising a plurality of reaction vessels, the reaction vessels being configured to receive and contain dissociated species therein.

65-81. -81. (canceled)

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

capturing a plurality of analyte molecules or particles on a first substrate;

releasing a plurality of molecules or particles from the first substrate;

detecting molecules or particles released from the first substrate on or within a second substrate comprising a plurality of reaction vessels; and

determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of molecules or particles released from the first substrate on or within the second substrate.

83-110. -110. (canceled)

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

capturing a plurality of analyte molecules or particles on a first substrate;

releasing a plurality of molecules or particles from the first substrate;

detecting molecules or particles released from the first substrate on or within a second substrate; and

determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of molecules or particles released from the first substrate, wherein the true concentration of the analyte molecules or particles in the fluid sample is less than about 50×

10<sup>−

15</sup> molar, and wherein the measure of the concentration determined in the determining act differs from the true concentration by no greater than 10%.

112-117. -117. (canceled)

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

capturing a plurality of analyte molecules or particles on a first substrate;

releasing a plurality of molecules or particles from the first substrate;

immobilizing at least one particle comprising a binding partner for the molecule or particle released from the first substrate to each of at least a fraction of the plurality of molecules or particles released from the first substrate to form a plurality of complexes;

detecting the plurality of complexes formed in the immobilizing act; and

determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of the complexes.

119-136. -136. (canceled)

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

capturing a plurality of analyte molecules or particles on a first substrate by immobilizing the plurality of analyte molecules or particles on the first substrate;

releasing a plurality of immobilized molecules or particles from the first substrate, wherein the number of molecules or particles released is related to the number of analyte molecules or particles captured;

detecting molecules or particles released from the first substrate on or within a second substrate, wherein at least a fraction of the molecules or particles released from the first substrate become immobilized with respect to the second substrate, and wherein the number of molecules or particles detected on or within the second substrate is no greater than the number of molecules or particles which are released from the first substrate; and

determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of molecules or particles released from the first substrate.

138-164. -164. (canceled)

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

capturing a plurality of analyte molecules or particles on a first substrate by immobilizing the plurality of analyte molecules or particles on the first substrate;

releasing a plurality of immobilized molecules or particles from the first substrate, wherein the number of molecules or particles released is related to the number of analyte molecules or particles captured;

immobilizing at least one first binding ligand with respect to each of at least a fraction of the plurality of analyte molecules or particles or to each of at least a fraction of the plurality of molecules or particles released from the first substrate;

capturing at least a fraction of the plurality of molecules or particles released from the first substrate on or within a second substrate;

exposing the second substrate to a plurality of precursor labeling agent molecules, wherein the plurality of precursor labeling agent molecules are converted to a plurality of labeling agent molecules upon exposure to first binding ligands; and

determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of the labeling agent molecules.

166-187. -187. (canceled)

188. A method of detecting analyte molecules or particles in a fluid sample, comprising:

exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component and at least one analyte molecule or particle;

dissociating at least a portion of each complex to form a plurality of dissociated species, which are not immobilized with respect to the substrate; and

detecting at least some of the plurality of the dissociated species substantially simultaneously, wherein substantially all of the detected dissociated species are spatially separated with respect to the other detected dissociated species during detection, such that detection is able to resolve individual dissociated species of the plurality of dissociated species.

189-199. -199. (canceled)