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

• US 20190302109A1
• Filed: 04/11/2019
• Published: 10/03/2019
• 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 11,619,631 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

1-141. -141. (canceled)

142. 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 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 a measure indicative of the number or fraction of said locations containing at least one analyte molecule or particle, wherein determining the measure indicative of the number or fraction of said locations containing at least one analyte molecule or particle comprises use of one or more detection methods comprising one or both of;

a) replicating a nucleic acid precursor, and

b) converting a nucleic acid precursor to a nucleic acid that can be readily detected; and

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

143. The method of claim 142, wherein the addressing step comprises determining the number of said locations containing at least one analyte molecule or particle.

144. The method of claim 142, wherein the addressing step comprises determining the fraction of said locations containing at least one analyte molecule or particle.

145. The method of claim 142, wherein the capture objects exposed to the solution each include a binding surface having affinity for at least one type of analyte molecule or particle.

146. The method of claim 145, wherein determining the measure indicative of the number or fraction of said locations containing at least one analyte molecule or particle comprises replicating a nucleic acid precursor.

147. The method of claim 145, wherein determining the measure indicative of the number or fraction of said locations containing at least one analyte molecule or particle comprises converting a nucleic acid precursor to a nucleic acid that can be readily detected.

148. The method of claim 145, wherein determining the measure indicative of the number or fraction of said locations containing at least one analyte molecule or particle further comprises determining a measure indicative of the number or fraction of locations comprising replicated nucleic acids.

149. The method of claim 145, wherein determining the measure indicative of the number or fraction of said locations containing at least one analyte molecule or particle further comprises determining a measure indicative of the number or fraction of locations comprising nucleic acids that can be readily detected.

150. The method of claim 145, wherein determining the measure indicative of the number or fraction of said locations containing at least one analyte molecule or particle comprises detecting a fluorescence signal.

152. The method of claim 145, wherein at least a portion of the analyte molecules or particles are associated with at least one binding ligand.

153. The method of claim 145, wherein determining the measure indicative of the number or fraction of said locations containing at least one analyte molecule or particle comprises using a nucleic acid-based technique chosen from one or more of:

polymerase chain reaction (PCR), rolling circle amplification (RCA), ligation, and loop-mediated isothermal amplification (LAMP).

154. The method of claim 145, 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.

155. The method of claim 145, wherein the plurality of locations comprises a plurality of reaction vessels.

156. The method of claim 145, wherein the analyte molecules or particles are proteins.

157. The method of claim 145, wherein the analyte molecules or particles are nucleic acids.

151. (canceled)

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

preparing a plurality of capture objects that each are associated with either at least one analyte molecule or particle of the fluid sample or are free of any analyte molecules or particles of the fluid sample;

individually isolating at least a portion of the capture objects subjected to the preparing step from each other;

individually addressing at least a portion of the capture objects individually isolated in the isolating step and determining a measure indicative of the number or fraction of the individually isolated capture objects associated with at least one analyte molecule or particle of the fluid sample; and

determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the measure indicative of the number or fraction of capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle of the fluid sample.

159. The method of claim 158, wherein the individually addressing step comprises determining the number of the individually isolated capture objects associated with at least one analyte molecule or particle of the fluid sample.

160. The method of claim 158, wherein the individually addressing step comprises determining the fraction of the individually isolated capture objects associated with at least one analyte molecule or particle of the fluid sample.

161. The method of claim 158, wherein the capture objects comprise or are each contained within a liquid droplet, and the step of individually isolating the at least a portion of the capture objects comprises suspending the liquid droplets in a fluid that is immiscible with the liquid droplets.

162-180. -180. (canceled)

181. 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;

detecting at each addressed location the presence or absence of a capture object and whether, if present, the capture object is associated with any of analyte molecules or particles; and

determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part by determining a measure indicative of the number or fraction of the capture objects subjected to the addressing step determined to be associated with at least one analyte molecule or particle.

182-185. -185. (canceled)