title: Fluorescent Biosensor for Phosphate Determination Based on Immobilized Polyfluorene-Liposomal Nanoparticles Coupled with Alkaline Phosphatase.
abstract: This work describes the development of a novel fluorescent biosensor based on the inhibition of alkaline phosphatase (ALP). The biosensor is composed of the enzyme ALP and the conjugated cationic polyfluorene HTMA-PFP. The working principle of the biosensor is based on the fluorescence quenching of this polyelectrolyte by p-nitrophenol (PNP), a product of the hydrolysis reaction of p-nitrophenyl phosphate (PNPP) catalyzed by ALP. Because HTMA-PFP forms unstable aggregates in buffer, with low fluorescence efficiency, previous stabilization of the polyelectrolyte was required before the development of the biosensor. HTMA-PFP was stabilized through its interaction with lipid vesicles to obtain stable blue-emitting nanoparticles (NPs). Fluorescent NPs were characterized, and the ability to be quenched by PNP was evaluated. These nanoparticles were coupled to ALP and entrapped in a sol-gel matrix to produce a biosensor that can serve as a screening platform to identify ALP inhibitors. The components of the biosensor were examined before and after sol-gel entrapment, and the biosensor was optimized to allow the determination of phosphate ion in aqueous medium.
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández , 03202, Elche, Alicante, Spain.
authors.forename: Zehra
authors.initials: Z
authors.lastname: Kahveci
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández , 03202, Elche, Alicante, Spain.
authors.forename: Maria José
authors.initials: MJ
authors.lastname: Martínez-Tomé
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández , 03202, Elche, Alicante, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: Mallavia
authors.affiliation: Instituto de Biología Molecular y Celular, Universidad Miguel Hernández , 03202, Elche, Alicante, Spain.
authors.forename: C Reyes
authors.initials: CR
authors.lastname: Mateo
mesh_terms: D000469:Alkaline Phosphatase; D015374:Biosensing Techniques; D005449:Fluorenes; D008081:Liposomes; D053758:Nanoparticles; D009596:Nitrophenols; D010710:Phosphates
chemical_list: D005449:Fluorenes; D008081:Liposomes; D009596:Nitrophenols; D010710:Phosphates; D000469:Alkaline Phosphatase; C024836:4-nitrophenol
keywords: alkaline phosphatase inhibitors; biosensor; conjugated polyelectrolytes; fluorescent nanoparticles; lipid vesicles; p-nitrophenol; phosphate ion