title: Construction of effective disposable biosensors for point of care testing of nitrite.
abstract: In this paper we aim to demonstrate, as a proof-of-concept, the feasibility of the mass production of effective point of care tests for nitrite quantification in environmental, food and clinical samples. Following our previous work on the development of third generation electrochemical biosensors based on the ammonia forming nitrite reductase (ccNiR), herein we reduced the size of the electrodes' system to a miniaturized format, solved the problem of oxygen interference and performed simple quantification assays in real samples. In particular, carbon paste screen printed electrodes (SPE) were coated with a ccNiR/carbon ink composite homogenized in organic solvents and cured at low temperatures. The biocompatibility of these chemical and thermal treatments was evaluated by cyclic voltammetry showing that the catalytic performance was higher with the combination acetone and a 40°C curing temperature. The successful incorporation of the protein in the carbon ink/solvent composite, while remaining catalytically competent, attests for ccNiR's robustness and suitability for application in screen printed based biosensors. Because the direct electrochemical reduction of molecular oxygen occurs when electroanalytical measurements are performed at the negative potentials required to activate ccNiR (ca.-0.4V vs Ag/AgCl), an oxygen scavenging system based on the coupling of glucose oxidase and catalase activities was successfully used. This enabled the quantification of nitrite in different samples (milk, water, plasma and urine) in a straightforward way and with small error (1-6%). The sensitivity of the biosensor towards nitrite reduction under optimized conditions was 0.55 A M(-1) cm(-2) with a linear response range 0.7-370 μM.
authors.affiliation: Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Instituto Superior de Ciências da Saúde Egas Moniz, Campus Universitário, Quinta da Granja, 2829-511 Caparica, Portugal; UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte Caparica, Portugal.
authors.forename: Tiago
authors.initials: T
authors.lastname: Monteiro
authors.affiliation: UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte Caparica, Portugal.
authors.forename: Patrícia R
authors.initials: PR
authors.lastname: Rodrigues
authors.affiliation: UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte Caparica, Portugal.
authors.forename: Ana Luisa
authors.initials: AL
authors.lastname: Gonçalves
authors.affiliation: UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte Caparica, Portugal.
authors.forename: José J G
authors.initials: JJ
authors.lastname: Moura
authors.affiliation: Sensors Unit, Materials Division, IK4-CIDETEC, Parque Tecnológico de San Sebastián, P° Miramón 196, 20009 Donostia - San Sebastián, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Jubete
authors.affiliation: Sensors Unit, Materials Division, IK4-CIDETEC, Parque Tecnológico de San Sebastián, P° Miramón 196, 20009 Donostia - San Sebastián, Spain.
authors.forename: Larraitz
authors.initials: L
authors.lastname: Añorga
authors.affiliation: Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Barbora
authors.initials: B
authors.lastname: Piknova
authors.affiliation: Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, United States.
authors.forename: Alan N
authors.initials: AN
authors.lastname: Schechter
authors.affiliation: UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte Caparica, Portugal; Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República - EAN, 2780-157 Oeiras, Portugal.
authors.forename: Célia M
authors.initials: CM
authors.lastname: Silveira
authors.affiliation: Centro de Investigação Interdisciplinar Egas Moniz (CiiEM), Instituto Superior de Ciências da Saúde Egas Moniz, Campus Universitário, Quinta da Granja, 2829-511 Caparica, Portugal; UCIBIO, REQUIMTE, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte Caparica, Portugal. Electronic address: mg.almeida@fct.unl.pt.
authors.forename: M Gabriela
authors.initials: MG
authors.lastname: Almeida
mesh_terms: D000096:Acetone / Q000737:chemistry; D000818:Animals; D015374:Biosensing Techniques; D002074:Butanones / Q000737:chemistry; D002374:Catalase / Q000737:chemistry; D002384:Catalysis; D045263:Cytochromes a1 / Q000737:chemistry*; D003575:Cytochromes c1 / Q000737:chemistry*; D060766:Drinking Water / Q000032:analysis; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D005949:Glucose Oxidase / Q000737:chemistry; D006108:Graphite / Q000737:chemistry*; D007281:Ink; D008892:Milk / Q000737:chemistry; D008904:Miniaturization; D009565:Nitrate Reductases / Q000737:chemistry*; D009573:Nitrites / Q000032:analysis* / Q000097:blood / Q000652:urine; D010100:Oxygen / Q000737:chemistry; D000067716:Point-of-Care Testing; D012997:Solvents / Q000737:chemistry
chemical_list: D002074:Butanones; D060766:Drinking Water; D004800:Enzymes, Immobilized; D009573:Nitrites; D012997:Solvents; D000096:Acetone; C005222:methylethyl ketone; D006108:Graphite; D045263:Cytochromes a1; D003575:Cytochromes c1; D005949:Glucose Oxidase; D002374:Catalase; D009565:Nitrate Reductases; C054690:nitrate reductase (cytochrome); D005947:Glucose; D010100:Oxygen
keywords: Biosensors; Carbon conductive ink; Cytochrome c nitrite reductase; Nitrite; Point of care testing; Screen printed electrodes