title: Sensitive bi-enzymatic biosensor based on polyphenoloxidases-gold nanoparticles-chitosan hybrid film-graphene doped carbon paste electrode for carbamates detection.
abstract: A bi-enzymatic biosensor (LACC-TYR-AuNPs-CS/GPE) for carbamates was prepared in a single step by electrodeposition of a hybrid film onto a graphene doped carbon paste electrode (GPE). Graphene and the gold nanoparticles (AuNPs) were morphologically characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, dynamic light scattering and laser Doppler velocimetry. The electrodeposited hybrid film was composed of laccase (LACC), tyrosinase (TYR) and AuNPs entrapped in a chitosan (CS) polymeric matrix. Experimental parameters, namely graphene redox state, AuNPs:CS ratio, enzymes concentration, pH and inhibition time were evaluated. LACC-TYR-AuNPs-CS/GPE exhibited an improved Michaelis-Menten kinetic constant (26.9±0.5M) when compared with LACC-AuNPs-CS/GPE (37.8±0.2M) and TYR-AuNPs-CS/GPE (52.3±0.4M). Using 4-aminophenol as substrate at pH5.5, the device presented wide linear ranges, low detection limits (1.68×10(-9)±1.18×10(-10)-2.15×10(-7)±3.41×10(-9)M), high accuracy, sensitivity (1.13×10(6)±8.11×10(4)-2.19×10(8)±2.51×10(7)%inhibitionM(-1)), repeatability (1.2-5.8% RSD), reproducibility (3.2-6.5% RSD) and stability (ca. twenty days) to determine carbaryl, formetanate hydrochloride, propoxur and ziram in citrus fruits based on their inhibitory capacity on the polyphenoloxidases activity. Recoveries at two fortified levels ranged from 93.8±0.3% (lemon) to 97.8±0.3% (orange). Glucose, citric acid and ascorbic acid do not interfere significantly in the electroanalysis. The proposed electroanalytical procedure can be a promising tool for food safety control.
authors.affiliation: REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Rua Dr. Bernardino de Almeida 431, 4200-072 Porto, Portugal; GELCORR, Dep. de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Bloco 940, Campus do Pici, 60455-970 Fortaleza, CE, Brazil.
authors.forename: Thiago M B F
authors.initials: TM
authors.lastname: Oliveira
authors.affiliation: REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Rua Dr. Bernardino de Almeida 431, 4200-072 Porto, Portugal; REQUIMTE, Dep. de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
authors.forename: M Fátima
authors.initials: MF
authors.lastname: Barroso
authors.affiliation: REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Rua Dr. Bernardino de Almeida 431, 4200-072 Porto, Portugal. Electronic address: sbm@isep.ipp.pt.
authors.forename: Simone
authors.initials: S
authors.lastname: Morais
authors.affiliation: REQUIMTE, Dep. de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.
authors.forename: Mariana
authors.initials: M
authors.lastname: Araújo
authors.affiliation: REQUIMTE, Dep. de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre s/n, 4169-007 Porto, Portugal.
authors.forename: Cristina
authors.initials: C
authors.lastname: Freire
authors.affiliation: GELCORR, Dep. de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Bloco 940, Campus do Pici, 60455-970 Fortaleza, CE, Brazil.
authors.forename: Pedro
authors.initials: P
authors.lastname: de Lima-Neto
authors.affiliation: GELCORR, Dep. de Química Analítica e Físico-Química, Centro de Ciências, Universidade Federal do Ceará, Bloco 940, Campus do Pici, 60455-970 Fortaleza, CE, Brazil.
authors.forename: Adriana N
authors.initials: AN
authors.lastname: Correia
authors.affiliation: REQUIMTE, Dep. de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal.
authors.forename: Maria B P P
authors.initials: MB
authors.lastname: Oliveira
authors.affiliation: REQUIMTE, Instituto Superior de Engenharia do Porto, Instituto Politécnico do Porto, Rua Dr. Bernardino de Almeida 431, 4200-072 Porto, Portugal.
authors.forename: Cristina
authors.initials: C
authors.lastname: Delerue-Matos
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002219:Carbamates / Q000032:analysis*; D002244:Carbon / Q000737:chemistry*; D048271:Chitosan / Q000737:chemistry*; D004566:Electrodes; D006046:Gold / Q000737:chemistry*; D006108:Graphite / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry*; D010084:Oxidation-Reduction; D010575:Pesticides / Q000032:analysis*; D056951:Photoelectron Spectroscopy; D012680:Sensitivity and Specificity; D013499:Surface Properties
chemical_list: D002219:Carbamates; D010575:Pesticides; D002244:Carbon; D006046:Gold; D006108:Graphite; D048271:Chitosan; D042845:Laccase; D014442:Monophenol Monooxygenase
keywords: Bi-enzymatic biosensor; Carbamates; Chitosan hybrid film; Gold nanoparticles; Graphene modified electrode