Quantum dot-encoded bead set for calibration and quantification of multiplexed assays, and methods for their use

A method of determining the concentration of at least one analyte in a multiplexed assay, the method comprising:

• providing a mixture comprising a first set of beads and a second set of beads, wherein the first set of beads comprise a first semiconductor nanocrystal and an oligonucleotide that selectively binds to the at least one analyte, and wherein the second set of beads comprise a second semiconductor nanocrystal and a biotinylated oligonucleotide;
• contacting the mixture with a sample suspected of containing the at least one analyte;
• contacting the mixture with streptavidin labeled with a third semiconductor nanocrystal;
• simultaneously imaging the mixture to produce an image, wherein the image comprises signal obtained from the first semiconductor nanocrystal, signal obtained from the second semiconductor nanocrystal, and signal obtained from the third semiconductor nanocrystal;
• preparing a titration curve from the signal obtained from the third semiconductor nanocrystal;
• and
• using the titration curve and the image to determine the concentration of the at least one analyte.

A method of preparing a titration curve, the method comprising:

• providing a set of control beads, wherein the set of control beads comprise a coding moiety and a calibration moiety;
• obtaining signal from the coding moiety;
• obtaining signal from the calibration moiety;
• and
• preparing a titration curve from the signal obtained from the calibration moiety.

A method of preparing a titration curve, the method comprising:

• providing a set of control beads, wherein the set of control beads comprise a first semiconductor nanocrystal and a biotinylated oligonucleotide;
• contacting the set with streptavidin labeled with a second semiconductor nanocrystal;
• simultaneously imaging the set to produce an image, wherein the image comprises signal obtained from the first semiconductor nanocrystal and signal obtained from the second semiconductor nanocrystal;
• and
• preparing a titration curve from the signal obtained from the second semiconductor nanocrystal.

A set of control beads comprising:

• a plurality of beads comprising a coding moiety and a calibration moiety, wherein:
• the coding moiety is present at a fixed concentration in the plurality of beads;
• and
• the calibration moiety is present at a range of concentrations in the plurality of beads.

A set of control beads comprising:

• a plurality of beads comprising a semiconductor nanocrystal and a biotinylated oligonucleotide, wherein:
• the semiconductor nanocrystal is present at a fixed concentration in the plurality of beads;
• and
• the biotinylated oligonucleotide is present at a range of concentrations in the plurality of beads.