A novel functionalisation process for glucose oxidase immobilisation in poly(methyl methacrylate) microchannels in a flow system for amperometric determinations

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 g...

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Veröffentlicht in:Talanta (Oxford) 2014-08, Vol.126, p.20-26
Hauptverfasser: Cerqueira, Marcos Rodrigues Facchini, Grasseschi, Daniel, Matos, Renato Camargo, Angnes, Lucio
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Grasseschi, Daniel
Matos, Renato Camargo
Angnes, Lucio
description 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 60samplesh−1, with detection and quantification limits of 0.50 and 1.66µmolL−1, respectively. Michaelis–Menten parameters (Vmax and KM) were calculated as 449±47.7nmolmin−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
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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 60samplesh−1, with detection and quantification limits of 0.50 and 1.66µmolL−1, respectively. Michaelis–Menten parameters (Vmax and KM) were calculated as 449±47.7nmolmin−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&lt;ttabled, 0.05, 4=2.78). 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The microreactor proposed here was able to work with high bioconversion and a frequency of 60samplesh−1, with detection and quantification limits of 0.50 and 1.66µmolL−1, respectively. Michaelis–Menten parameters (Vmax and KM) were calculated as 449±47.7nmolmin−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&lt;ttabled, 0.05, 4=2.78). 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The microreactor proposed here was able to work with high bioconversion and a frequency of 60samplesh−1, with detection and quantification limits of 0.50 and 1.66µmolL−1, respectively. Michaelis–Menten parameters (Vmax and KM) were calculated as 449±47.7nmolmin−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&lt;ttabled, 0.05, 4=2.78). 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source MEDLINE; Elsevier ScienceDirect Journals Complete
subjects Amperometry
Dimethyl Sulfoxide - chemistry
Electrochemical Techniques - instrumentation
Electrochemical Techniques - methods
Enzymes
Enzymes, Immobilized - chemistry
Enzymes, Immobilized - metabolism
Glucose
Glucose - chemistry
Glucose - metabolism
Glucose oxidase
Glucose Oxidase - chemistry
Glucose Oxidase - metabolism
Glutaral - chemistry
Hydrogen Peroxide - chemistry
Hydrogen Peroxide - metabolism
Kinetics
Methodology
Microchannels
Microreactor
Oxidation-Reduction
Poly(methyl methacrylate)
Polyethyleneimine - chemistry
Polymethyl Methacrylate - chemistry
Polymethyl methacrylates
Reproducibility of Results
Samples
Spectrophotometry - methods
Statistical analysis
Statistical methods
title A novel functionalisation process for glucose oxidase immobilisation in poly(methyl methacrylate) microchannels in a flow system for amperometric determinations
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