High precision and cost-effective multiplex quantification of ab40, ab42, p181tau, p217tau, nfl, and gfap from plasma and serum

A bioassay system for multiplexed detection and quantification of multiple analytes in a biological sample, the bioassay system comprising:

• a plurality of sets of color-coded microspheres, the plurality of sets of color-coded microspheres including a first set of microspheres distinguishable by a first unique color code generated by internal dyes, each microsphere in the first set being coated with a specific capture agent that binds to Aβ40, a second set of microspheres distinguishable by a second unique color code generated by internal dyes, each microsphere in the second set being coated with a specific capture agent that binds to Aβ42, a third set of microspheres distinguishable by a third unique color code generated by internal dyes, each microsphere in the third set being coated with a specific capture agent that binds to p217Tau, a fourth set of microspheres distinguishable by a fourth unique color code generated by internal dyes, each microsphere in the fourth set being coated with a specific capture agent that binds to GFAP, and a fifth set of microspheres distinguishable by a fifth unique color code generated by internal dyes, each microsphere in the fifth set being coated with a specific capture agent that binds to Neurofilament Light Chain (NFL);
• a fluidic system configured to mix the biological sample with the plurality of sets of color-coded microspheres to allow for specific binding between analytes and their corresponding capture agent among other analytes and their respective capture agents, and to introduce reporter molecules that bind to captured analytes, the reporter molecules configured to emit a detectable reporter fluorescence upon excitation;
• and
• a detection system comprising a flow cell and capable of exciting and reading fluorescence of each internal dye and the detectable reporter fluorescence indicative of analyte binding,
• wherein the detection system is configured to identify each of the sets of microspheres by its associated unique color code and quantify bound analytes based on the detectable reporter fluorescence, alongside quantification of other analytes.

A method for multiplexed detection and quantification of multiple analytes in a biological sample, the method comprising:

• mixing the biological sample with a plurality of sets of color-coded microspheres, the plurality of sets of color-coded microspheres including a first set of microspheres distinguishable by a first unique color code generated by internal dyes, each microsphere in the first set being coated with a specific capture agent that binds to Aβ40, a second set of microspheres distinguishable by a second unique color code generated by internal dyes, each microsphere in the second set being coated with a specific capture agent that binds to Aβ42, a third set of microspheres distinguishable by a third unique color code generated by internal dyes, each microsphere in the third set being coated with a specific capture agent that binds to p181Tau or p217Tau, a fourth set of microspheres distinguishable by a fourth unique color code generated by internal dyes, each microsphere in the fourth set being coated with a specific capture agent that binds to GFAP, and a fifth set of microspheres distinguishable by a fifth unique color code generated by internal dyes, each microsphere in the fifth set being coated with a specific capture agent that binds to Neurofilament Light Chain (NFL);
• allowing specific binding between analytes in the biological sample and their corresponding capture agents on the microspheres;
• introducing reporter molecules that bind to captured analytes, the reporter molecules configured to emit a detectable reporter fluorescence upon excitation;
• passing the microspheres through a flow cell;
• exciting and reading fluorescence of each internal dye and the detectable reporter fluorescence indicative of analyte binding;
• and
• identifying each of the sets of microspheres by its associated unique color code and quantifying bound analytes based on the detectable reporter fluorescence.