Apparatus and methods using tethered enzymes for the detection of the enzymatic activity of biomarkers

A coupled enzyme reaction point of care assay system for detection and measurement of an active enzyme biomarker present in a sample of the blood of a patient, the system comprising:

• a blood collection vial including pre-treatment materials adapted to remove anti-oxidants from a collected blood sample, the materials selected from at least one of the group including:
• uric acid and uricase, ascorbate oxidase and ascorbic acid, or glucose oxidase and glucose;
• a point of care blood collector for collecting a patient's blood into a blood collection vial;
• a biomarker detection mechanism comprising at least three fluid flow entities, each fluid flow entity having a fluid input zone and a corresponding reaction zone, and a blood insertion member for placing a preset amount of said blood from the blood collection vial onto each of said fluid input zones, each fluid flow entity adapted to provide for the flow of fluid from each fluid input zone to the corresponding reaction zone, at least one fluid flow entity being a test strip, at least one fluid flow entity being a negative control strip and at least one fluid flow entity being a positive control strip, the test strip and positive control strip each including a band located between the fluid input zone and the reaction zone, the band comprising a multiplicity one or more substrates with which the active enzyme biomarker will react, the test strip and positive control strip being adapted to facilitate the fluid flowing from each strip's fluid input zone to its reaction zone to carry the one or more substrates in the band into the corresponding reaction zone, each reaction zone of each fluid flow entity comprising a multiplicity of components, said components comprising:
• (i) a multiplicity of one or more types of substrates, co-factors, buffers and cryoprotectants, (ii) a multiplicity of at least two types of tethered enzymes each of the at least two types of tethered enzymes comprising at least 100 enzyme molecules tethered to a nanoparticle, each of the at least 100 enzyme molecules of the first type of tethered enzymes adapted to react to a reaction product formed from a reaction between the biomarker with the one or more other substrates in the band where present,
• the positive control strip also including a pre-set quantity of the enzyme biomarker;
• and
• a photonic luminescence reader adapted to measure an amplitude of light emitted from each of the reaction zones, the photonic luminescence reader including electronics adapted to calculate a measurement of an active enzyme biomarker present in the sample of the blood of a patient from the measurements of light amplitude from the reaction zones.