title: A novel functionalisation process for glucose oxidase immobilisation in poly(methyl methacrylate) microchannels in a flow system for amperometric determinations.
abstract: Different materials like glass, silicon and poly(methyl methacrylate) (PMMA) are being used to immobilise enzymes in microchannels. PMMA shows advantages such as its low price, biocompatibility and attractive mechanical and chemical properties. Despite this, the introduction of reactive functional groups on PMMA is still problematic, either because of the complex chemistry or extended reaction time involved. In this paper, a new methodology was developed to immobilise glucose oxidase (GOx) in PMMA microchannels, with the benefit of a rapid immobilisation process and a very simple route. The new procedure involves only two steps, based on the reaction of 5.0% (w/w) polyethyleneimine (PEI) with PMMA in a dimethyl sulphoxide medium, followed by the immobilisation of glucose oxidase using a solution containing 100U enzymes and 1.0% (v/v) glutaraldehyde. The reactors prepared in this way were evaluated by a flowing system with amperometric detection (+0.60V) based on the oxidation of the H2O2 produced by the reactor. The microreactor proposed here was able to work with high bioconversion and a frequency of 60 samples h(-1), with detection and quantification limits of 0.50 and 1.66µmol L(-1), respectively. Michaelis-Menten parameters (Vmax and KM) were calculated as 449±47.7nmol min(-1) and 7.79±0.98mmol. Statistical evaluations were done to validate the proposed methodology. The content of glucose in natural and commercial coconut water samples was evaluated using the developed method. Comparison with spectrophotometric measurements showed that both methodologies have a very good correlation (tcalculated, 0.05, 4=1.35<ttabled, 0.05, 4=2.78).
authors.affiliation: Departamento de Química, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, MG 36036-900, Brazil; Universidade de São Paulo, Instituto de Química, Avenida Professor Lineu Prestes, 748, São Paulo, SP CEP 05508-000, Brazil.
authors.forename: Marcos Rodrigues Facchini
authors.initials: MR
authors.lastname: Cerqueira
authors.affiliation: Universidade de São Paulo, Instituto de Química, Avenida Professor Lineu Prestes, 748, São Paulo, SP CEP 05508-000, Brazil.
authors.forename: Daniel
authors.initials: D
authors.lastname: Grasseschi
authors.affiliation: Departamento de Química, Universidade Federal de Juiz de Fora (UFJF), Juiz de Fora, MG 36036-900, Brazil.
authors.forename: Renato Camargo
authors.initials: RC
authors.lastname: Matos
authors.affiliation: Universidade de São Paulo, Instituto de Química, Avenida Professor Lineu Prestes, 748, São Paulo, SP CEP 05508-000, Brazil. Electronic address: luangnes@iq.usp.br.
authors.forename: Lucio
authors.initials: L
authors.lastname: Angnes
mesh_terms: D004121:Dimethyl Sulfoxide / Q000737:chemistry; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005949:Glucose Oxidase / Q000737:chemistry* / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D010084:Oxidation-Reduction; D011094:Polyethyleneimine / Q000737:chemistry; D019904:Polymethyl Methacrylate / Q000737:chemistry*; D015203:Reproducibility of Results; D013053:Spectrophotometry / Q000379:methods
chemical_list: D004800:Enzymes, Immobilized; D011094:Polyethyleneimine; D019904:Polymethyl Methacrylate; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D005947:Glucose; D005976:Glutaral; D004121:Dimethyl Sulfoxide
keywords: Amperometry; Glucose; Glucose oxidase; Microreactor; Poly(methyl methacrylate)