Methods for reducing noise in signal-generating digital assays

A method for reducing fluorescence background noise in a signal-generating digital assay used to detect an analyte in a sample, the method comprising:

• (a) contacting a fluid sample containing or suspected of containing the analyte with a plurality of solid supports to create a mixture, wherein each solid support comprises one or more first specific binding member capable of binding to the analyte thereby producing solid support-first specific binding member-analyte complexes;
• (b) washing the solid support-first specific binding member-analyte complexes in the mixture with a first wash buffer to remove any solid support-first specific binding member not bound to the analyte thereby producing a washed mixture;
• (c) adding to the washed mixture one or more second specific binding members capable of binding to the analyte thereby producing solid support-first specific binding member-analyte-second specific binding member complexes, wherein the second specific binding member comprises a signal generating compound attached thereto;
• (d) washing the solid support-first specific binding member-analyte-second specific binding member complexes with a second wash buffer to remove any second specific binding member not bound to the analyte bound to the first specific binding member thereby producing washed solid support complexes;
• (e) adding to the washed solid support complexes a signal generating substrate, wherein the signal generating compound and the signal generating substrate produce a detectable signal;
• (f) spatially segregating at least a portion of the washed solid support complexes into a plurality of separate locations, wherein the plurality of separation locations comprises a plurality of reaction vessels and wherein the washed solid support complexes are present in an aqueous phase;
• (g) covering the plurality of reaction vessels with a sealant, wherein the aqueous phase is displaced by the sealant in the reaction chamber;
• and
• (h) detecting the presence or absence of the detectable signal using a digital counting device, wherein detection of the presence of the detectable signal indicates the presence of a single molecule of analyte in the sample,
• wherein the digital counting device comprises a black device.

A method for reducing fluorescence background noise in a fluorescent digital immunoassay used to detect an analyte in a sample, the method comprising:

• (a) contacting a fluid sample containing or suspected of containing the analyte with a plurality of solid supports to create a mixture, wherein each solid support comprises one or more first specific binding member capable of binding to the analyte thereby producing solid support-first specific binding member-analyte complexes;
• (b) washing the solid support-first specific binding member-analyte complexes in the mixture with a first wash buffer to remove any solid support-first specific binding member not bound to the analyte thereby producing a washed mixture;
• (c) adding to the washed mixture one or more second specific binding members capable of binding to the analyte thereby producing solid support-first specific binding member-analyte-second specific binding member complexes, wherein the second specific binding member comprises a signal generating compound attached thereto;
• (d) washing the solid support-first specific binding member-analyte-second specific binding member complexes with a second wash buffer to remove any second specific binding member not bound to the analyte bound to the first specific binding member thereby producing washed solid support complexes;
• (e) adding to the washed solid support complexes a signal generating substrate, wherein the signal generating compound and the signal generating substrate produce a detectable signal;
• (f) spatially segregating at least a portion of the washed solid support complexes into a plurality of separate locations, wherein the plurality of separation locations comprises a plurality of reaction vessels and wherein the washed solid support complexes are present in an aqueous phase;
• (g) covering the plurality of reaction vessels with a sealant, wherein the aqueous phase is displaced by the sealant in the reaction chamber;
• and
• (h) detecting the presence or absence of the detectable signal using a digital counting device, wherein detection of the presence of the detectable signal indicates the presence of a single molecule of analyte in the sample,
• wherein the digital counting device comprises a black device.