title: A novel combined thermometric and amperometric biosensor for lactose determination based on immobilised cellobiose dehydrogenase.
abstract: A novel method for lactose determination in milk is proposed. It is based on oxidation of lactose by cellobiose dehydrogenase (CDH) from the basidiomycete Phanerochaete chrysosporium, immobilised in an enzyme reactor. The reactor was prepared by cross-linking CDH onto aminopropyl-silanised controlled pore glass (CPG) beads using glutaraldehyde. The combined biosensor worked in flow injection analysis (FIA) mode and was developed for simultaneous monitoring of the thermometric signal associated with the enzymatic oxidation of lactose using p-benzoquinone as electron acceptor and the electrochemically generated current associated with the oxidation of the hydroquinone formed. A highly reproducible linear response for lactose was obtained between 0.05 mM and 30 mM. For a set of more than 500 samples an R.S.D. of less than 10% was achieved. The assay time was ca. 2 min per sample. The sensor was applied for the determination of lactose in dairy milk samples (milk with a fat content of 1.5% or 3% and also "lactose free" milk). No sample preparation except dilution with buffer was needed. The proposed method is rapid, suitable for repeated use and allows the possibility to compare results from two different detection methods, thus providing a built-in quality assurance. Some differences in the response observed between the methods indicate that the dual approach can be useful in mechanistic studies of redox enzymes. In addition, a dual system opens up interesting possibilities for studies of enzyme properties and mechanisms.
authors.affiliation: Department of Biochemistry and Structural Biology, Lund University, PO Box 124, 22100 Lund, Sweden. Maria.Yakovleva@gmail.com
authors.forename: Maria
authors.initials: M
authors.lastname: Yakovleva
authors.forename: Orsolya
authors.initials: O
authors.lastname: Buzas
authors.forename: Hirotoshi
authors.initials: H
authors.lastname: Matsumura
authors.forename: Masahiro
authors.initials: M
authors.lastname: Samejima
authors.forename: Kiyohiko
authors.initials: K
authors.lastname: Igarashi
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Larsson
authors.forename: Lo
authors.initials: L
authors.lastname: Gorton
authors.forename: Bengt
authors.initials: B
authors.lastname: Danielsson
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000295:instrumentation*; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry*; D002417:Cattle; D003217:Conductometry / Q000295:instrumentation*; D004800:Enzymes, Immobilized / Q000737:chemistry; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005504:Food Analysis / Q000295:instrumentation*; D007785:Lactose / Q000032:analysis*; D008892:Milk / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018511:Systems Integration; D013817:Thermography / Q000295:instrumentation*
chemical_list: D004800:Enzymes, Immobilized; D002237:Carbohydrate Dehydrogenases; C019859:cellobiose-quinone oxidoreductase; D007785:Lactose