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
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12235267-B2
Publication Date
2025-02-25
Expiration Date
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
The invention provides a method for determining a measure of the concentration of analyte molecules or particles in a fluid sample by using capture objects that include both capture objects that are associated with at least one type of analyte molecule or particle and capture objects that are not associated with that analyte type. The method passes at least some of the capture objects by a detection system and individually interrogates the capture objects with the detection system during passage, then determines a measure indicative of the number or fraction of the capture objects passed that are associated with the at least one analyte type.
The concentration measure is then determined based at least in part on the measure indicative of the number or fraction of associated capture objects. The approach enables ultra-sensitive analyte detection and quantification by reducing non-specific binding background, where only a statistically significant fraction of capture objects becomes associated with the target analyte while others remain unbound.
In one described framework, the capture objects are spatially segregated into many individually addressable locations, including sealed reaction vessel arrays or single-file microfluidic droplets, and analyte association is counted in an on/off manner across the locations, with optional intensity-based readout. The document also describes use of an optical detection system, including a light source and a detector such as a CCD or a fiber optic array.
Claims Coverage
The independent claim covers an analyte concentration determination method that infers analyte concentration from a fraction or number of capture objects that are associated with an analyte type after individual interrogation by a detection system. The inventive features include four merged elements: using capture objects that are partly associated and partly not associated, passing and individually interrogating the capture objects, determining a measure indicative of the associated capture objects, and determining concentration based on that measure.
Capture objects with associated and unassociated fractions
Providing capture objects, wherein at least some of the capture objects are associated with at least one type of analyte molecule or particle from the fluid sample and at least some of the capture objects are not associated with the at least one type of analyte molecule or particle.
Passing and individual interrogation by detection system
Passing at least some of the capture objects by a detection system and individually interrogating with the detection system the at least some of the capture objects during the passing.
Measure indicative of associated capture object number or fraction
Determining, based at least in part on step (c), a measure indicative of the number or fraction of the capture objects passed by the detection system that are associated with the at least one type of analyte molecule or particle.
Concentration determination based on associated capture object measure
Determining a measure of the concentration of the at least one type of analyte molecule or particle in the fluid sample based at least in part on the measure indicative of the number or fraction of the capture objects passed by the detection system that are associated with the at least one type of analyte molecule or particle.
Overall, the claim coverage is centered on inferring analyte concentration from the measured number or fraction of capture objects that become associated with an analyte type, after those capture objects are passed and individually interrogated by a detection system.
Stated Advantages
Enables ultra-sensitive analyte detection and quantification by reducing non-specific binding background.
Concentration determination is supported across regimes by selecting between binary on/off read-out and analog intensity read-out, with calibration curves mapping measured fractions or intensities to known concentrations.
Quantitation robustness is supported by using statistical analysis such as Poisson distribution analysis (and optionally Gaussian/Poisson approaches) tied to the fraction of active locations.
Improved quantitation sensitivity is discussed, including reported limit of detection (LOD) and sensitivity gains associated with example embodiments using enzymatic labeling for proteins and labeling for DNA.
Documented Applications
Analyte concentration determination in a fluid sample using capture objects passed through and individually interrogated by a detection system.
Spatially segregated detection in sealed reaction vessel arrays or single-file microfluidic droplets with binary on/off counting of individually addressable locations, with optional intensity readout.
Use with an optical detection system including a light source and detectors such as CCD or fiber optic arrays to detect interrogation results.
Nucleic-acid amplification labeling options for analyte-associated detectable signals, including PCR, RCA, ligation, and LAMP.
Quantifying concentrations of protein analytes such as PSA and TNF-α in a fluid sample using capture objects, detection system interrogation, and fraction/intensity-based calibration/quantitation, including discussion of enzymatic labeling-based examples (e.g., sandwich assay context).
Quantifying concentrations of DNA in a fluid sample using capture objects, detection interrogation, and calibration/quantitation with statistical analysis, including discussion of signaling and calibration/quantitation strategy using arrays of reaction locations.
Performing quantitation in a partitioned detection format using many discrete locations (e.g., femtoliter reaction wells and beads arrays) where binary on/off counting or analog intensity read-out is used to infer analyte concentration from the fraction of positive/active locations.
Interested in licensing this patent?