Apparatus and methods using coupled enzyme luminescence assays for the detection of analytes and biomarkers

A coupled tethered enzyme luminescence assay adapted to measure the amount of enzymatic activity of neuron specific enolase in a liquid sample derived from the blood of a patient comprising:

• at least one test well and at least one positive control well, each comprising a freeze-dried mixture of a plurality of components, the at least one positive control well including a first layer and a second layer separately introduced into each positive control well during assay preparation, the test well including only a single layer having the same plurality of components as the first layer of the at least one positive control well;
• the first layer of the positive control well comprising 2-phosphoglycerate, an ADK inhibitor, a plurality of first tethered enzyme nanobots, each first tethered enzyme nanobot formed by tethering at least one hundred pyruvate kinase enzyme to a silica nanoparticle and a plurality of second tethered nanobots, each second tethered enzyme nanobot formed by tethering at least one hundred luciferase enzyme molecules to a silica nanoparticle, the tethering being structured to provide oriented immobilization of the tethered enzymes pyruvate kinase and luciferase;
• each of the pyruvate kinase and luciferase enzymes having two different types of affinity tags comprising a first type of affinity tag adapted to facilitate extraction of enzymes from a liquid and a second type of affinity tag adapted to provide tethering to a silica nanoparticle;
• the second layer of the at least one positive control well including a preset amount of an enolase enzyme.

A coupled tethered enzyme luminescence assay adapted to measure the amount of enzymatic activity of neuron specific enolase in a liquid sample derived from the blood of a patient comprising:

• at least one test well and at least one positive control well, each comprising a freeze-dried mixture of a plurality of components;
• the test well comprising 2-phosphoglycerate, an ADK inhibitor, a plurality of first tethered enzyme nanobots, each of the first tethered enzyme nanobots, each first tethered enzyme nanobot formed by tethering at least one hundred pyruvate kinase enzyme to a silica nanoparticle and a plurality of second tethered nanobots, each second tethered enzyme nanobot formed by tethering at least one hundred luciferase enzyme molecules to a silica nanoparticle, the tethering being structured to provide oriented immobilization of the tethered enzymes pyruvate kinase and luciferase;
• each of the pyruvate kinase and luciferase enzymes having two different types of affinity tags comprising a first type of affinity tag being a 6xhis tag adapted to facilitate extraction of enzymes from a liquid and a second type of affinity tag being an SiO2 tag adapted to provide tethering to a silica nanoparticle;
• the positive control well, including all the components of the at least one test well with the addition of a preset amount of an enolase enzyme.