Ultra-sensitive detection of molecules using dual detection methods

A method for determining a measure of the concentration of analyte molecules or particles in a fluid sample, comprising:

• providing the analyte molecules or particles immobilized with respect to a binding surface having affinity for at least one type of analyte molecule or particle, the binding surface forming or contained within one of a plurality of locations on a substrate, such that at least some of the plurality of locations contain at least one analyte molecule or particle and a statistically significant fraction of the plurality of locations do not contain any analyte molecules or particles;
• exposing the analyte molecules or particles to a first type of binding ligand having an affinity for a first epitope on the analyte molecules or particles and a second type of binding ligand having an affinity for a second epitope on the analyte molecules or particles;
• addressing at least a portion of the plurality of the locations and determining the number of said locations containing a first type of binding ligand and a second type of binding ligand;
• and
• determining a measure of the concentration of analyte molecules or particles in the fluid sample based at least in part on the number of locations containing both a first type of binding ligand and a second type of binding ligand.