Ultra-sensitive detection of molecules or particles using beads or other capture objects

Inventors

Duffy, David C. • Rissin, David M. • Walt, David R. • Fournier, David • Kan, Cheuk

Assignees

Quanterix Corp

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.

Core Innovation

A method for determining a measure of a concentration of analyte molecules or particles in a fluid sample is provided by exposing a plurality of capture objects to a solution containing or suspected of containing analyte, and immobilizing analyte with respect to the capture objects such that at least some capture objects associate with analyte and a statistically significant fraction of capture objects do not associate with any analyte molecule or particle. The method spatially segregates at least a portion of the capture objects into a plurality of separate locations.

A measure indicative of a number or fraction of locations containing at least one analyte molecule or particle is determined by one or both of replicating a nucleic acid precursor and converting a nucleic acid precursor to a nucleic acid that can be readily detected. The concentration of analyte molecules or particles is determined based at least in part on the measure indicative of the number or fraction of locations containing at least one analyte molecule or particle.

The disclosed quantification framework includes calibration between a binary determination of positive locations and analyte concentration, including calibration relationships that can be linear or non-linear. For low-concentration regimes, the method uses Poisson distribution analysis on the number or fraction of locations containing analyte, and the method combines digital and analog analysis through a common calibration curve.

Claims Coverage

The independent claim covers a partitioned capture-object format with immobilization and spatial segregation into separate locations, followed by determining analyte concentration from a measure of how many locations are positive. The claim includes nucleic-acid precursor replication and/or conversion to readily detectable nucleic acids as detection methods, and concentration determination based on the number or fraction of positive locations.

Across the independent claim and its refinements, the core claim coverage requires immobilization of analyte with a statistically significant negative fraction, partitioning into separate locations, and inferring analyte concentration from a number or fraction of positive locations determined via nucleic-acid precursor replication and/or conversion to readily detectable nucleic acids. Dependent refinements further specify Poisson distribution analysis, nucleic-acid detection technique choices, and fluorescence signal detection.

Stated Advantages

Documented Applications