Immobilization of Firefly Luciferase on PVA-co-PE Nanofibers Membrane as Biosensor for Bioluminescent Detection of ATP

Abstract

The bioluminescent reaction catalyzed by firefly luciferase has become widely established as an outstanding analytical system for assay of adenosine triphosphate (ATP). When in solution, the luciferase is unstable and cannot be reused. The problem can be partially solved by immobilizing the luciferase on solid substrates. The poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibers membrane has abundant active hydroxyl groups on the surface. The PVA-co-PE nanofibers membrane was first activated by cyanuric chloride with triazinyl group. Then the activated PVA-co-PE nanofibers membrane was subsequently reacted with 1,3-propanediamine and biotin. The firefly luciferase was immobilized onto the surface of 1,3-propanediamine- and biotin-functionalized membranes. The surface chemical structure and morphologies of nanofibers membranes were characterized by FTIR-ATR spectra and SEM. The hydrophilicity of membranes was tested by water contact angle measurements. The detection of fluorescence intensity displayed that the firefly-luciferase-immobilized PVA-co-PE nanofibers membranes indicated high catalytic activity and efficiency. Especially, the firefly-luciferase-immobilized nanofiber membrane which was functionalized by biotin can be a promising candidate as biosensor for bioluminescent detection of ATP because of its high detection sensitivity.

Firefly Luciferase Mutant with Enhanced Activity and Thermostability

“… 12 Alternatively, instead of engineering the enzyme, synthetic luciferin substrates have been developed that improve cell permeability of the substrate to improve emission and alter the luminescence spectra of the emitted light. 13 − 15 Recombinant luciferases have also been used for applications in vitro, for example, as biosensors 16 and as the light source in bioluminescence resonance energy transfer reactions. 17 , 18 Such applications will also benefit from engineered luciferases with improved sensitivity and thermostability.…”

Efficient adsorption of dyes from aqueous solution by poly(vinyl alcohol‐co‐ethylene) nanofibre membranes modified with β‐cyclodextrin

“…Materials PVA-co-PE nanofibre membranes were prepared according to reported methods [33][34][35]. b-CD was purchased from Aladdin (Shanghai, China, analytical pure).…”

Integration of Nanomaterials and Bioluminescence Resonance Energy Transfer Techniques for Sensing Biomolecules

“…Similar to Lux, Fluc is ATP-dependent and requires the presence of co-factors, oxygen and magnesium, in order to complete its reaction with D-luciferin [44,45,46]. Since its discovery, Fluc has been used in various fields as a biosensor through recombination with another protein of interest and as ATP sensor, taking advantage of its ATP-dependency [47,48,49]. Fluc demonstrates high light quantum yield making it superior in comparison to Lux and aequorin [45].…”