Enzymatic amplification of a flow-injected thermometric enzyme-linked immunoassay for human insulin

A flow-injected thermometric enzyme linked immunoassay for human insulin which employs the lactate dehydrogenase/lactate oxidase (LDH/LOD) substrate recycling system for signal amplification is described. The system is composed of two columns, an immunosorbent column containing immobilized anti-insulin antibodies for sensing and a recycling column containing immobilized LDH/LOD/Catalase for detection. The effect of flow rates, conjugate concentrations, and chromatographic support material upon the sensitivity of the assay are investigated. The assay has a detection limit of 0.025 microgram/ml and a linear range from 0.05 to 2 micrograms/ml. This corresponds to a 10-fold increase in sensitivity over the unamplified system. A recombinant human insulin-proinsulin conjugate was also tested. The results show that enzymatic amplification can be employed to increase the sensitivity and reproducibility of flow injection assay-based biosensors. The implications of these results upon on-line analysis are discussed.