Detection of analyte
AssigneeNorthwestern University
InventorsKeith E.J. Tyo, Catherine Elizabeth Majors
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Abstract
A method for detecting an analyte is described in which the simultaneously binding of two fusion proteins (i.e., a sandwich assay in solution) is used, bringing two halves of a split enzyme together to produce product, which is detected via a FRET-based biosensor. The method may incorporate an autocatalytic feedback loop that responds to enzymatic product by producing more product to provide ultrasensitive, bistable detection of analyte that is tunable over several orders of magnitude. This system is broadly applicable for protein and small molecule detection.
Claims18
A method of detecting an analyte comprising:
- adding to a sample comprising adenosine triphosphate and an unknown concentration of analyte detection reagents comprising a first reagent and a second reagent, the first reagent comprises a first protein bound to a first fragment of a first CAMP catalyzing enzyme;
- the second reagent comprises a second protein bound to a second fragment of the first CAMP catalyzing enzyme, the first protein and the second protein are configured to form with the analyte a protein-analyte-protein complex, the first fragment of the first CAMP catalyzing enzyme and the second fragment of the first cAMP catalyzing enzyme together restitute the full first CAMP catalyzing enzyme;
- and
- then measuring a cAMP concentration in the sample and determining the concentration of the analyte from the measured cAMP concentration, wherein the analyte is rapamycin, one of the first protein and the second protein is FKBP12-rapamycin-binding (FRB) protein and the other of the first protein and the second protein is FK506 (Tacrolimus)-Binding Protein 12 (FKBP12).
A kit for determining a concentration of an analyte, comprising a first reagent comprises a first protein bound to a first fragment of a first CAMP catalyzing enzyme;
- and
- a second reagent comprises a second protein bound to a second fragment of the first CAMP catalyzing enzyme, the first protein and the second protein are configured to form with the analyte a protein-analyte-protein complex, the first fragment of the first CAMP catalyzing enzyme and the second fragment of the first CAMP catalyzing enzyme together restitute the full first CAMP catalyzing enzyme, wherein the analyte is rapamycin, one of the first protein and the second protein is FKBP12-rapamycin-binding (FRB) protein and the other of the first protein and the second protein is FK506 (Tacrolimus)-Binding Protein 12 (FKBP12).
Dependencies are inferred from the claim text.