Nucleic acid cleaving enzyme-based biosensor and methods of use thereof

A biosensor for detection of an analyte comprising:

• a) i) a nucleic acid substrate coupled to a first support and a nucleic acid cleaving enzyme;
• or
• ii) a nucleic acid cleaving enzyme comprising a nucleic acid substrate coupled to a first support;
• and
• b) a nucleic acid binding molecule coupled to a microzone on a second support;
• wherein the nucleic acid substrate comprises a detection moiety;
• wherein the nucleic acid enzyme is configured for cleaving the nucleic acid substrate;
• wherein the nucleic acid cleaving enzyme is configured to be activated upon contact with the analyte, and the activated nucleic acid cleaving enzyme is configured for cleaving the nucleic acid substrate to release a cleavage product comprising the detection moiety;
• wherein the nucleic acid binding molecule is configured for binding to the cleavage product,
• wherein the nucleic acid substrate coupled to the first support or the nucleic acid cleaving enzyme comprising a nucleic acid substrate coupled to the first support, and the nucleic acid binding molecule coupled to a microzone on the second support, are configured for use in a container, optionally a test tube, and
• wherein the biosensor is configured to be used with a test solution of up to about 100 mL comprising the analyte.