title: Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing.
abstract: In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd(1) (cyt-cd(1)) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c(552) (cyt-c(552)), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 μM cyt-cd(1)/100 μM cyt-c(552) and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c(552) and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254±2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c(552) and cd(1) is 9.9×10(3)M(-1)s(-1). The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 μM; detection and quantification limits of 7 and 24 μM, respectively; sensitivity of 2.49±0.08 Amol(-1)cm(2) μM(-1). Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.
authors.affiliation: REQUIMTE - Dept. de Química, CQFB, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: A S
authors.initials: AS
authors.lastname: Serra
authors.forename: S R
authors.initials: SR
authors.lastname: Jorge
authors.forename: C M
authors.initials: CM
authors.lastname: Silveira
authors.forename: J J G
authors.initials: JJ
authors.lastname: Moura
authors.forename: E
authors.initials: E
authors.lastname: Jubete
authors.forename: E
authors.initials: E
authors.lastname: Ochoteco
authors.forename: G
authors.initials: G
authors.lastname: Cabañero
authors.forename: H
authors.initials: H
authors.lastname: Grande
authors.forename: M G
authors.initials: MG
authors.lastname: Almeida
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism; D003580:Cytochromes / Q000737:chemistry* / Q000378:metabolism; D055664:Electrochemical Techniques / Q000379:methods; D004566:Electrodes; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D053520:Marinobacter / Q000201:enzymology; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism; D009573:Nitrites / Q000032:analysis*; D010084:Oxidation-Reduction; D058105:Polymerization; D011142:Polyvinyl Alcohol / Q000737:chemistry; D013696:Temperature
chemical_list: D003574:Cytochrome c Group; D003580:Cytochromes; D009573:Nitrites; D002244:Carbon; D011142:Polyvinyl Alcohol; C022302:cytochrome C-552; D009572:Nitrite Reductases; C028123:nitrite reductase (NO-forming)