Methods and systems for extending dynamic range in assays for the detection of molecules or particles
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.
1. A system for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:
at least one detector configured to address locations of an assay substrate, able to produce at least one signal indicative of the presence or absence of an analyte molecule or particle at each location addressed and having an intensity varying with the number of analyte molecules or particles at each location; and
at least one signal processor configured to determine from the at least one signal a measure indicative of the number or percentage of said locations containing at least one analyte molecule or particle, and further configured to, based upon the measure indicative of the number or percentage, either determine 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 percentage of locations containing at least one analyte molecule or particle, or determine a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on an intensity level of the at least one signal indicative of the presence of a plurality of analyte molecules or particles.
2. The system of claim 1 , wherein when the percentage of said locations containing at least one analyte molecule or particle is less than about 40%, less than about 35%, or less than about 30%, the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on the measure indicative of the number or percentage of locations containing at least one analyte molecule or particle.
3. The system of claim 1 , wherein when the percentage of said locations containing at least one analyte molecule or particle is less than about 80%, less than about 75%, less than about 70%, less than about 65%, or less than about 60%, the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on the measure indicative of the number or percentage of locations containing at least one analyte molecule or particle.
4. The system of claim 1 , wherein when the percentage of said locations containing at least one analyte molecule or particle is greater than about 30%, greater than about 35%, greater than about 40%, or greater than about 45%, the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on an intensity level of the at least one signal indicative of the presence of a plurality of analyte molecules or particles.
5. The system of claim 1 , wherein when the percentage of said locations containing at least one analyte molecule or particle is greater than about 60%, greater than about 65%, greater than about 70%, or greater than about 75%, the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on an intensity level of the at least one signal indicative of the presence of a plurality of analyte molecules or particles.
6. The system of claim 1 , wherein when the percentage of said locations containing at least one analyte molecule or particle is between about 30% and about 50%, or between about 35% and about 45%, or about 40%, the measure of the concentration of analyte molecules or particles in the fluid sample is an average of the 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 percentage of locations containing at least one analyte molecule or particle and the measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on an intensity level of the at least one signal indicative of the presence of a plurality of analyte molecules or particles.
7. The system of claim 1 , wherein when the percentage of said locations containing at least one analyte molecule or particle is between about 60% and about 80%, or between about 65% and about 75%, or about 70%, the measure of the concentration of analyte molecules or particles in the fluid sample is an average of the 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 percentage of locations containing at least one analyte molecule or particle and the measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on an intensity level of the at least one signal indicative of the presence of a plurality of analyte molecules or particles.
8. The system of claim 1 , further comprising the assay substrate operatively coupled to the at least one detector.
9. The system of claim 8 , wherein the locations of the assay substrate each comprise a binding surface forming or contained within such locations.
10. The system of claim 9 , wherein at least one binding surface comprises at least one analyte molecule or particle immobilized on the binding surface.
11. The system of claim 9 , wherein the binding surface is contained within such locations.
12. The system of claim 11 , wherein the binding surface comprises a capture object.
13. The system of claim 12 , wherein the capture object comprises a bead.
14. The system of claim 12 , wherein the locations addressed are locations which contain at least one capture object.
15. The system of claim 1 , wherein the at least one signal processor is configured to determine from the at least one signal a measure indicative of the number of said locations containing at least one analyte molecule or particle, and is further configured to, based upon the measure indicative of the number, either determine 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 of locations containing at least one analyte molecule or particle, or determine a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on an intensity level of the at least one signal indicative of the presence of a plurality of analyte molecules or particles.
16. The system of claim 1 , wherein the at least one signal processor is configured to determine from the at least one signal a measure indicative of the percentage of said locations containing at least one analyte molecule or particle, and is further configured to, based upon the measure indicative of the percentage, either determine 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 percentage of locations containing at least one analyte molecule or particle, or determine a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on an intensity level of the at least one signal indicative of the presence of a plurality of analyte molecules or particles.
17. The method of claim 1 , wherein the at least one signal processor is configured such that when the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on the measure indicative of the number of locations containing at least one analyte molecule or particle, the measure of the concentration is based at least in part on a ratio of the number of said locations associated with at least one analyte molecule or particle of the fluid sample to the number of locations not associated with an analyte molecule or particle of the fluid sample.
18. The method of claim 17 , wherein the locations addressed are locations which are determined to contain at least one capture object.
19. The method of claim 1 , wherein the at least one signal processor is configured such that when the measure of the concentration of analyte molecules or particles in the fluid sample is based at least in part on the measure indicative of the number of locations containing at least one analyte molecule or particle, the measure of the concentration is based at least in part on a Poisson distribution analysis involving the number of the locations associated with at least one analyte molecule or particle of the fluid sample.
20. The method of claim 1 , wherein the locations are reaction vessels each having a volume between about 10 attoliters and 100 picoliters.