Efficient bienzyme nanocomposite film for chiral recognition of L-tryptophan, L-phenylalanine and L-tyrosine

An efficient bienzyme nanocomposite film-based biosensor for L-amino acid recognition has been developed and characterized. The biosensor was fabricated by immobilizing horseradish peroxidase (HRP) and L-amino acid oxidase (L-AAODx) in poly(toluidine blue) film (polyTB) at the surface of a multi-walled carbon nanotube (CNTs) modified glassy carbon electrode (GCE). Only L-amino acids can be electrochemically oxidized by L-AAODx in the presence of oxygen (O-2) to produce hydrogen peroxide (H2O2), which can be further reduced to H2O by HRP at a low potential of -0.1 V. The concentration of L-amino acids was estimated by the reduction current of H2O2. The polyTB film, acting as the enzyme-immobilization carrier and redox mediator, accelerated electron transfer between analytes and the electrode. At the same time, CNTs with excellent conductivity and strong adsorption ability enhanced the utilization of the enzymes. The performance of this biosensor was evaluated by electrochemical techniques and it showed high selectivity and fast response for the chiral recognition of L-amino acids, including L-tryptophan (L-Trp), L-phenylalanine (L-Phe) and L-tyrosine (L-Tyr). The low potential reduced the interference of some coexisting biomolecules for the determination of the L-amino acids. The recovery of the three amino acids in milk indicated the feasibility of this biosensor for use in complex samples.