Ultra-sensitive detection of enzymes by capture-and-release followed by quantification

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

• exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component and at least one analyte molecule or particle;
• exposing the plurality of complexes to a plurality of binding ligands comprising an enzymatic component, wherein at least one binding ligand is immobilized with respect to at least some of the complexes;
• exposing the plurality of complexes to a dissociating agent, which has essentially no specific affinity for the capture components, to dissociate at least a portion of at least some of the plurality of molecules or particles from the first substrate, thereby forming a plurality of dissociated species, wherein at least some of the plurality of dissociated species comprise at least a portion of the enzymatic component;
• detecting at least some of the plurality of dissociated species;
• and
• determining a measure of the concentration of the analyte molecules or particles in the fluid sample based on the detection of the plurality of dissociated species.

A method of detecting analyte molecules or particles in a fluid sample, comprising:

• exposing a substrate comprising a plurality of capture components to a sample comprising a plurality of analyte molecules or particles, so that analyte molecules or particles associate with capture components to form a plurality of complexes, each complex being immobilized with respect to the substrate and comprising at least one capture component, at least one analyte molecule or particle, and at least one enzymatic component;
• exposing the substrate to a dissociating agent to cause at least a portion of at least some of the plurality of complexes to dissociate from the substrate to form a plurality of dissociated species, which are not immobilized with respect to the substrate and wherein at least some of the dissociated species each comprise at least a portion of the enzymatic component;
• and
• detecting a plurality of the dissociated species substantially simultaneously, wherein each of the detected dissociated species are spatially separated with respect to the other detected dissociated species during detection, such that detection is able to resolve individual dissociated species of the plurality of dissociated species.