Optimization of flow-injection systems for determination of substrates by means of enzyme amplification reactions and chemiluminescence detection

A flow-injection system is described that incorporates a small column reactor containing two co-immobilized, synergistically operating oxidoreductases, allowing determination of minute amounts of substrates by means of enzyme amplification and subsequent chemiluminescence detection of the hydrogen peroxide generated in the repeated redox cycling. With lactate oxidase and lactate dehydrogenase, and taking advantage of the fact that the enzymatic degradation step and the ensuing detection step can be individually optimized, the FIA-system has been optimized by factorial experiments to yield an amplification factor of over 140 for each of the two substrates lactate and pyruvate. With a linear calibration range of 0-6muM, the limits of detection for the two species were 48 and 103nM, respectively, and the sampling rate was 50-60/hr. The optimized system has also been employed for assay of glucose by utilizing a column reactor with immobilized glucose oxidase and glucose dehydrogenase, but yielded amplification factors of only 3-4. The large discrepancy in the performance of the two enzyme systems is discussed.