A transient enzymatic inhibition as an efficient tool for the discriminating bioluminescent analysis of three adenylic nucleotides with a fiberoptic sensor based on a compartmentalized tri-enzymatic sensing layer

A fiberoptic biosensor has been developed for the specific and alternate determination of ATP, ADP and AMP. The sensing layer is arranged by compartmentalizing the tri-enzyme sequence adenylate kinase – creatine kinase – firefly luciferase. The two kinases are covalently co-immobilized on a collagen membrane, whereas firefly luciferase is bound alone on a separate one. For the specific determination of each adenylic nucleotide, three particular reaction media are needed with which flow-injection analysis can be performed in the 2.5–2500 pmol for ATP, 10–2500 pmol for ADP and 25–5000 pmol for AMP linear ranges. For ten consecutive injections of 100 pmol ATP, 100 and 250 pmol ADP, the RSD were 3.5, 2.0 and 2.2%, respectively. When the three nucleotides are present simultaneously in the same sample, the transient inhibition of adenylate-kinase activity by adenosine 5′-monosulphate enables their specific and alternate measurement.