title: Recombinant laccase: II. Medical biosensor.
abstract: Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.22 nm, compressibility coefficient 37.5 m/N). The sensitivity of the biosensor was investigated via UV spectroscopy, and linearity was found in the absorbance peak shift at 400 nm at drug concentration varying up to 20 uM. The enzyme kinetics was subsequently investigated with potentiometric and amperometric measurements, and we found electronic transfer of at least 1 electron, k(s) 0.57 s(-1), diffusion coefficient 3 × 10(-6) cm(2)/s, K(cat) 6825.92 min(-1), K(M) 4.1 uM, K(cat)/K(M) 2.8 × 10(7) mol(-1) s(-1), sensitivity of 440 nA/uM, maximum velocity 1706.48 nA/s, and response time less than 5 s. The amperometric and potentiometric measurements were repeated after a month, confirming the stability of the biosensor.
authors.affiliation: Biophysics and Nanobiotechnology Laboratories, Department of Experimental Medicine, University of Genova, Genoa 16121-16167, Italy.
authors.forename: Nicola Luigi
authors.initials: NL
authors.lastname: Bragazzi
authors.forename: Eugenia
authors.initials: E
authors.lastname: Pechkova
authors.forename: Dora
authors.initials: D
authors.lastname: Scudieri
authors.forename: Tercio Bezerra Correia
authors.initials: TB
authors.lastname: Terencio
authors.forename: Manuela
authors.initials: M
authors.lastname: Adami
authors.forename: Claudio
authors.initials: C
authors.lastname: Nicolini
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002997:Clomipramine / Q000097:blood / Q000737:chemistry; D004566:Electrodes; D006801:Humans; D007700:Kinetics; D042845:Laccase / Q000737:chemistry* / Q000235:genetics; D018625:Microscopy, Atomic Force / Q000379:methods; D011199:Potentiometry / Q000295:instrumentation; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: D011994:Recombinant Proteins; D042845:Laccase; D002997:Clomipramine