Study On A Fiber Optic Glucose Biosensor Based On Fluorescence Quenching

Fiber optic biosensing technology is a novel interdiscipline, it has a wide application prospect in medical science, biological engineering, food industries and environment monitoring. As for the determination of D-glucose in the field of clinical diagnosis and preclinical medicine, especially in the diagnosis of diabetic patients, a glucose sensor is considered to be very desirable. The existing traditional methods for the determination of glucose have such deficiencies as low sensitivity or slow response that can't meet the need of practical use. Therefore, it's necessary to develop a method for fast determination of glucose.In this thesis, we have done plenty of experiments on developing a fiber optic glucose biosensor based on fluorescence quenching to realize fast detection of glucose. Our main works are:1. The catalytic performance of free glucose oxidase(GOD) was studied and some basic properties of GOD such as best pH for catalytic reaction, best buffer concentration and optimium temperature were acquired.2. Some substrates including cellulose actate, micro glassbeads/optical fiber, sol-gel system for GOD immobilization were studied and the performance of the immobilized GOD were compared. Results showed that cellulose actate membrane can meet the needs of future fiber optic glucose sensor applications.3.Systematic research was done on the preparation of CA membranes for GOD immobilization. CA membranes were prepared by dumping and salt leaching methods and GOD were immobilized on these membranes. Performance of immobilized GOD showed that CA membranes prepared by salt leaching had better performance but lower rate of finished products, which means that this methods can not be put into mass-production but only for laboratory uses.4.The structure of CA membranes, immobilization and reacting conditions are the influencing factors of the activity of immobilized GOD, Covalent binding and covalent-coupling methods were employed and the effects of activity of GOD membranes were contrasted. The whole immobilizing process and was studied and optimized including effects of activating conditions, glutaraldehyde (GA), albumin of bovine serum (BSA), GOD loading,etc. Results showed thatcovalent-coupling is the best way to immobilize GOD.5.We devised a fiber optic glucose biosensor based on fluorescence quenching, cellulose acetate (CA) was chosen as substrate and Ru(bPY)3Cl2 as fluorescence dye. Fluorescence was born by exited light from LED (416nm) via bifurcated fiber optic bundles reaching the sensor head which was immerged in glucose solution. Then the life time of fluorescence which was dynamically quenched by molecular oxygen was measured with a novel "lock-in phase amplifying" technology .The performance of the sensor was investigated including response(less than 30s), analytical range(50 mg/dl to 500 mg/dl), repeatability,best pH range(between 6.0 to 7.0) and lifetime of the sensor(at least 10 weeks),etc. Repeatability tests showed that the relative deviation of this glucose sensor is 5.9%, which turned out to be desirable for preclinical use.