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
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Publication Number
US-10725032-B2
Publication Date
2020-07-28
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 determines a measure of the concentration of analyte molecules or particles in a fluid sample based on the fraction of capture objects associated with at least one analyte molecule or particle. At least a portion of the capture objects are individually addressed, and the measured fraction is used to determine concentration. A digital readout counts positive and negative capture objects, with a Poisson distribution adjustment or a calibration curve used to estimate concentration.
The independent claim specifies that the capture objects comprise or are contained within liquid droplets that are immiscible in the suspending fluid during at least the addressing step. The droplets flow through a flow channel so that the capture objects pass by a detection system. The description also combines digital counting at low occupancy with intensity-based readout at higher occupancy to extend dynamic range.
The drawings and description report bead-based digital immunoassay arrangements in which analyte concentration corresponds to the fraction of positive capture beads or to fluorescence or chemiluminescence signals. Reported analytes and assay components include streptavidin-β-galactosidase, PSA, TNF-α, DNA, and alkaline phosphatase. Optional embodiments mention enzymatic labels, calibration, and Poisson adjustment.
Claims Coverage
The supplied claims identify one independent claim, claim 1. Its coverage is summarized in three inventive features concerning capture objects associated with analyte, concentration determination from the associated fraction, and addressing in immiscible droplets that flow past a detection system.
Capture objects associated with analyte
Capture objects are provided, each associated with at least one analyte molecule or particle in the fluid sample or free of analyte molecules or particles.
Individually addressing capture objects and determining concentration from the associated fraction
At least a portion of the capture objects are individually addressed, the fraction associated with at least one analyte molecule or particle is determined, and a measure of the concentration of analyte molecules or particles in the fluid sample is determined based at least in part on that fraction.
Capture objects in immiscible droplets passing a detection system
The capture objects comprise or are each contained within a liquid droplet that is immiscible within the suspending fluid during at least the addressing step, and at least a portion flow through a flow channel and pass by a detection system.
Claim 1 covers determining analyte concentration from the fraction of capture objects associated with analyte, where the capture objects are individually addressed in immiscible droplets flowing through a flow channel past a detection system.
Stated Advantages
Combining digital counting at low occupancy with intensity-based readout at higher occupancy extends dynamic range.
The reported single-enzyme detection reaches 220 zM, compared with 15 fM by plate-reader ensemble measurement.
The description reports attomolar detection of PSA, TNF-α, and DNA.
The described approach is intended to reduce the effects of nonspecific background.
The approach is intended to enable detection at very low analyte concentrations.
Documented Applications
Determining a measure of the concentration of analyte molecules or particles in a fluid sample, including bead-based measurements involving streptavidin-β-galactosidase, PSA, TNF-α, DNA, and alkaline phosphatase.
Detection of PSA, TNF-α, and DNA is described.
PSA detection is reported in all 30 tested post-prostatectomy patient samples.
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