Biofunctionalized and bioluminescent PVA membrane for ATP detection

This work demonstrates the suitability of biofunctionalized membranes to exhibit bioluminescent properties and proves their selectivity, stability, and reusability. This is demonstrated by fabricating a transparent polyvinyl alcohol (PVA) membrane functionalized with a firefly luciferase enzyme. This enzyme catalyzes a light-producing reaction that involves adenosine triphosphate (ATP), a key molecule present in all living organisms, and therefore a universal biomarker for microbial contamination in food, water, and surfaces. It is known that the detection of ATP by free luciferase is effective. However, the free enzyme tends to deactivate rapidly, cannot be easily reused, its shelf life is not constant, and it is relatively expensive. To overcome these challenges, firefly luciferase was immobilized on polyvinyl alcohol (PVA) membrane, a biodegradable and transparent material that enhances signal clarity. The characterization of the biofunctionalized membrane by ATR-FTIR spectroscopy, confocal microscopy, and circular dichroism elucidated the attachment, distribution, and structural changes of the enzyme loaded into the membrane. The work provided direct evidence for the influence of the membrane structural parameters on the distribution of the enzyme on the membrane and its bioluminescent activity. Bioluminescence activity confirmed the good sensitivity of the system to ATP (1·10−11 mol/L) and the reusability of the enzyme over four consecutive reaction cycles. Besides, the results demonstrated the robustness and selectivity of the bioluminescent PVA membrane in real-world streams, such as seawater, and in water desalinated by direct contact membrane distillation (DCMD), highlighting the potential of bioluminescent membranes for the development of practical biosensors for simplified and complex streams.