NOVEL ENZYME IMMOBILIZATION FOR BIOSENSOR APPLICATIONS (BACTERIA)

ENZYME CRYSTALS, CROSS-LINKED USING A BIFUNCTIONAL REAGENT SUCH AS GLUTARALDEHYDE, CONSTITUTE A NOVEL FORM OF ENZYME IMMOBILIZATION. SUCH CROSS-LINKED IMMOBILIZED ENZYME CRYSTALS (CLIECS) ARE SUPERIOR TO CONVEN- TIONALLY IMMOBILIZED ENZYMES IN MANY RESPECTS. FOR EXAMPLE, THE CRYSTAL LATTICE INTERACTIONS, SUPPLEMENTED BY CHEMICAL CROSS-LINKS, PROVIDE THE INDIVIDUAL ENZYME MOLECULES WITHIN A CLIEC WITH AN ENHANCED STABILITY THAT ALLOWS FOR FUNCTION IN HARSH ENVIRONMENTS (SUCH AS ELEVATED TEMPERATURES AND UNUSUAL SOLVENTS) THAT WOULD NOT OTHERWISE BE COMPATIBLE WITH CATALYTIC ACTIVITY. THESE FEATURES OF CLIECS LEND THEMSELVES TO BIOSENSOR APPLICATIONS, WHERE IT IS DESIRABLE TO PRODUCE THE LARGEST POSSIBLE SIGNAL FROM THE SMALLEST POSSIBLE VOLUME OF CATALYST AND QUANTITY OF SUBSTRATE. IN THE PHASE I STUDY, CLIECS OF BACTERIAL LUCIFERASE FROM "VIBRIO HARVEYI" WILL BE INVESTIGATED IN A PROTOTYPE SYSTEM TO DEMONSTRATE THE FEASIBILITY OF THIS APPROACH TO BIOSENSOR APPLICATIONS. LUCIFERASE IS CHOSEN BECAUSE IT PRODUCES LIGHT AS CONSEQUENCE OF ITS CATALYTIC ACTIVITY. THE EXCEPTIONAL SENSITIVITY AND EFFICIENCY OF PHOTON SENSING DEVICES CAN BE EXPLOITED TO DETECT FEMTOMOLAR CONCENTRATIONS OF SELECTED ANALYTES UNDER APPROPRIATE EXPERIMENTAL CONDITIONS. A BROAD ARRAY OF CLINICALLY INTERESTING ENZYMATIC REACTIONS HAVE BEEN DESCRIBED IN THE LITERATURE THAT CAN BE READILY COUPLED TO LIGHT PRODUCTION BY A LUCIFERASE CLIEC.