A biosensor based on immobilization of lactate oxidase in a PB-CTAB film for FIA determination of lactate in beer samples

Abstract

No presente trabalho são apresentados resultados referentes à fabricação de um biossensor para análise de lactato. O dispositivo foi preparado imobilizando-se a enzima lactato oxidase em filme de Azul da Prússia (PB, sigla em inglês) depositado na superfície de eletrodo de carbono vítreo em soluções de CTAB. Nestas condições experimentais observou-se melhoria na sensibilidade e seletividade das determinações de lactato, as quais foram conduzidas em sistema FIA com detecção amperométrica. Nas condições otimizadas (pH = 6,9 e E = −0,1 V), obteve-se curva analítica com limite superior igual a 0,28 mmol L -1 e um limite de detecção de 0,84 µmol L -1 . A repetibilidade do método foi avaliada por meio de injeções consecutivas de amostras de lactato 0,28 mmol L -1 , obtendo-se desvio padrão de 2,2 % (n = 18). O método foi aplicado em amostras de cerveja e houve boa concordância entre os resultados obtidos e aqueles oriundos do uso de procedimento espectrofotométrico de referência.An amperometric lactate biosensor with lactate oxidase immobilized into a Prussian Blue (PB) modified electrode was fabricated. The advantage of using cetyltrimethylammonium bromide (CTAB) in the electrodeposition step of PB films onto glassy carbon surfaces was confirmed taking into account both the stability and sensitivity of the measurements. The biosensor was used in the development of a FIA amperometric method for the determination of lactate. Under optimal operating conditions (pH = 6.9, E = −0.1 V), the linear response of the method was extended up to 0.28 mmol L -1 lactate with a limit of detection of 0.84 µmol L -1 . The repeatability of the method for injections of a 0.28 mmol L -1 lactate solution was 2.2 % (n = 18). The usefulness of the method was demonstrated by determining lactate in beer samples and the results were in good agreement with those obtained by using a reference spectrophotometric enzyme method.Keywords: lactate, biosensor, prussian blue modified electrodes, CTAB, FIA amperometric detection, beer IntroductionThe relevance of metal hexacyanoferrates for electroanalytical chemistry was firstly demonstrated in the pioneering studies of Itaya et al. 1,2 involving the electrocatalysis of oxygen and hydrogen peroxide reduction at surfaces covered by Prussian Blue (PB, iron(III) hexacyanoferrate(II)) films. The transfer of electrons and the movement of small hydrated counter-cations through the channels and holes of the solid for charge compensation is a typical feature of this kind of compound. Owing to the zeolite nature of metal hexacyanoferrates, their ability as effective three-dimensional electrocatalysts has been extensively recognized.Once sufficient negative potentials are applied to PB modified electrodes, electrons are injected into the film and the so-called Prussian-White film is generated which arises from the reduction of a high-spin Fe 3+ . The reduced form (Fe 2+ ) is catalytically active towards the electron-transfer step involving hydrogen peroxide, water being formed as a final product. At positive pot...

A self-powered amperometric lactate biosensor based on lactate oxidase immobilized in dimethylferrocene-modified LPEI

“…These results are consistent with the reported literature on the activity of free LOx in solution, which indicates that the FcMe2-LPEI polymer used to immobilize the enzyme does not significantly interfere with its characteristic activity. (Lowinsohn and Bertotti 2008) Additionally, it should be noted that under physiological conditions (pH 7.4 and 37 ºC), the electrode film is within 85% of its maximum activity with respect to both temperature and pH. The ability to maintain activity under these conditions is crucial when considering the possibility of using FcMe2-LPEI/LOx as an implantable sensor.…”

Improved reuse and storage performances at room temperature of a new environmentally friendly lactate oxidase biosensor prepared by ambient electrospray immobilization

“…Electrochemical experiments were carried out at room temperature by amperometric analysis with an applied potential of −0.10 V vs. the Ag/AgCl reference electrode. 59 The study of the LOX activity versus l -lactic acid was performed in a total volume of 100 μL of 0.1 M PBS buffer at pH 7, recording the current signals every 0.5 s. The fabricated biosensor must catalyze the oxidation of the substrate l -lactate into pyruvate. This reaction occurs in the presence of dissolved oxygen that oxidizes LOX by producing hydrogen peroxide which in turn is reduced by Prussian blue, which is finally reduced at the electrode surface (see Fig.…”

Current State of Sensors and Sensing Systems Utilized in Beer Analysis

“…An amperometric sensor was also developed with lactate dehydrogenase which was immobilized into a Prussian blue-modified electrode for the detection of lactate with a LOD of 0.84 µM. The lactate detection in beer with this method performed better as compared with other commercial kits [118].…”