Methods and systems for extending dynamic range in assays for the detection of molecules or particles
Inventors
Rissin, David M. • Fournier, David • Duffy, David C.
Assignees
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Publication Number
US-9551663-B2
Publication Date
2017-01-24
Expiration Date
Abstract
Described herein are systems and methods for extending the dynamic range of assay methods and systems used for determining the concentration of analyte molecules or particles in a fluid sample. In some embodiments, a method comprises spatially segregating a plurality of analyte molecules in a fluid sample into a plurality of locations. At least a portion of the locations may be addressed to determine the percentage of said locations containing at least one analyte molecule. Based at least in part on the percentage, a measure of the concentration of analyte molecules in the fluid sample may be determined using an analog, intensity-based detection/analysis method/system and/or a digital detection/analysis method/system. In some cases, the assay may comprise the use of a plurality of capture objects.
Core Innovation
The invention determines a measure of the concentration of analyte molecules or particles in a fluid sample by measuring the percentage of substrate locations containing at least one analyte. Based on that percentage, concentration is determined using the number of occupied locations or the measured intensity of a signal indicative of multiple analyte molecules or particles. Digital occupancy counting with Poisson correction is used at lower concentrations, while intensity-based analog analysis is used at higher concentrations.
The described approach addresses the limited measurement range of analog-only analysis by linking digital and analog regimes with a calibration factor and calibration curve. The reported combined dynamic range exceeds 6 logs, including an experiment reporting about 6.5 logs, compared with about 3 logs for analog-only plate-reader measurements. The document also describes determining analyte concentration using individually addressed capture objects and the percentage associated with analyte.
Claims Coverage
The two independent claims cover two inventive features for determining analyte concentration in a fluid sample: one based on analyte-containing substrate locations and one based on capture objects.
Concentration determination using substrate locations
Claim 1 provides analyte molecules or particles immobilized with respect to a binding surface forming or contained within substrate locations, determines the percentage of locations containing at least one analyte, and uses that percentage to determine concentration based on occupied-location count or measured signal intensity.
Concentration determination using capture objects
Claim 21 provides capture objects that are either associated with at least one analyte molecule or particle or are free of analyte, determines the percentage associated with analyte, and uses that percentage to determine concentration based on the number of locations containing analyte-associated capture objects or measured signal intensity.
The independent claims cover concentration measurement through occupancy percentages, using either substrate locations with binding surfaces or individually addressed capture objects, and permit concentration determination from occupied-location counts or signal intensity.
Stated Advantages
The combined digital and analog approach provides a dynamic range exceeding 6 logs; one reported experiment reaches about 6.5 logs, compared with about 3 logs for analog-only plate-reader measurements.
Documented Applications
Determining a measure of the concentration of analyte molecules or particles in a fluid sample.
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