A novel homogeneous electrogenerated chemiluminescence immunoassay (ECLIA) for the determination of small hapten was developed. As a model system, digoxin was investigated while luminol served as luminescence label and BSA served as carrier protein. Digoxin was indirectly heavily labeled with luminol throughBSAto form luminol-BSA-digoxin conjugate. The immunocomplex of luminol-BSA-digoxin conjugate with anti-digoxin antibody underwent less ECL reaction than luminol-BSA-digoxin conjugate after immunoreaction took place. Two immunoassay formats, directly homogeneous immunodetection for anti-digoxin antibody and competitive immunoassay for digoxin, were proposed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration was determined in the range from 1/50,000 to 1/2000 dilution. The ECL intensity versus digoxin concentration was linear in the range from 5.0×10−10 to 3.0×0−8 gml−1. The detection limit was 2.8×10−10 gml−1. The relative standard derivation for 1.0×10−9 gml−1 was 5.1%. The proposed method has been applied to assay digoxin in human control serum with satisfactory results.

A novel disposable electrochemical capillary-fill device for glucose was fabricated using thick-film technology and incorporating a water-soluble layer bearing Os-complex mediator and glucose oxidase. A simple dual-electrode system used as a transducer consisted of three carbon stripes for a working electrode and an Ag/AgCl strip for a reference/counter electrode. The reagent ink, containing glucose oxidase, osmium-Tris(4,7-dimethyl-1,10-phenanthroline), and water-soluble binder, was drop-coated on the biosensor base material between the strips of the carbon electrode. The electrode part was covered with thin glass cover in order to construct a microliter electrochemical capillary-fill. A drop of test solution is automatically introduced to the microchannel by capillary action and then a water-soluble layer bearing glucose oxidase, the mediator and buffer salts becomes solubilized and quickly dispersed throughout the volume of the microchannel. The composition of the enzyme and mediator in the water-soluble layer was optimized. The constructed device showed a wide linear response range for glucose from 0.1 to 25 mM, fast response, and good storage stability. The present investigation showed that an efficient biosensor was constructed with an extremely low loading of enzyme and mediator.

A novel flow injection analytical system with a screen-printed enzyme sensor incorporating Os-complex mediator has been developed. A newly created glucose biosensor was fabricated using thick-film technique and co-immobilization of glucose oxidase and Os-complex on a permeable membrane. The composition of the enzyme and mediator in the membrane and the analytical conditions were optimized. The flow injection electrochemical system showed that glucose was detected linearly in the concentration range from 0.1 to 10 mMwith a detection limit of 0.03 mMby 50ml of sample injection, giving a throughout of about 40 samples per hour. The glucose biosensors retained their constant response after more than 100 injections and storage over a month. The designed electrochemical flow system with a disposable biosensor is suitable for automatic and rapid determination of glucose.

A series of novel substituted-1, 10-phenanthroline complexes of osmium and ruthenium have been synthesized and characterized electrochemically with respect to their ability to act as electron transfer mediator for glucose oxidase. These complexes exhibit high second-order rate constants for reduced glucose oxidase in the order of 106-107M−1 s-1. The complexes bearing electron-donating substituents give a significantly higher rate constant with glucose oxidase and lower redox potential than those bearing electron-accepting substituents. 4, 7-Dimethy-1, 10-phenanthroline and 3, 4, 7, 8-tetramethy-1, 10-phenanthroline complexes of osmium have been characterized in details as mediator for glucose oxidase. The application of the family of mediators in amperometric biosensors seems feasible.f