Development of a novel reagentless, screen-printed amperometric biosensor based on glutamate dehydrogenase and NAD+, integrated with multi-walled carbon nanotubes for the determination of glutamate in food and clinical applications

© 2015 Elsevier B.V. Abstract A screen printed carbon electrode (SPCE) containing the electrocatalyst Meldola's Blue (MB) has been investigated as the base transducer for a reagentless glutamate biosensor. The biopolymer chitosan (CHIT) and multiwalled carbon nanotubes (MWCNTs) were used to encapsulate the enzyme glutamate dehydrogenase (GLDH) and the co-factor nicotinamide adenine dinucleotide (NAD+). The biosensor was fabricated by sequentially depositing the components on the surface of the transducer (MB-SPCE) in a layer-by-layer process, details of which are included in the paper. Each layer was optimised to construct the reagentless device. The biosensor was used in conjunction with amperometry in stirred solution using an applied potential of +0.1 V (vs. Ag/AgCl). Optimum conditions for the analysis of glutamate were found to be: temperature, 35°C; phosphate buffer, pH 7 (0.75 mM, containing 0.05 M NaCl). The linear range of the reagentless biosensor was found to be 7.5-105 μM, and limit of detection was found to be 3 μM (based on n = 5, CV: 8.5% based on three times signal to noise) and the sensitivity was 0.39 nA/μM (±0.025, coefficient of variation (CV) of 6.37%, n = 5). The response time of the biosensor was 20-30 s. A food sample was analysed for monosodium glutamate (MSG). The endogenous content of MSG was 90.56 mg/g with a CV of 7.52%. The reagentless biosensor was also used to measure glutamate in serum. The endogenous concentration of glutamate was found to be 1.44 mM (n = 5), CV: 8.54%. The recovery of glutamate in fortified serum was 104% (n = 5), CV of 2.91%

Inhibited enzymatic reaction of crosslinked lactate oxidase through a pH-dependent mechanism

Lactate oxidase (LOx), recognized to selectively catalyze the lactate oxidation in complex matrices, has been highlighted as preferable biorecognition element for the development of lactate biosensors. In a previous work, we have demonstrated that LOx crosslinking on a modified screen-printed electrode results in a dual range lactate biosensor, with one of the analysis linear range (4 to 50 mM) compatible with lactate sweat levels. It was advanced that such behavior results from an atypical substrate inhibition process. To understand such inhibition phenomena, this work relies in the study of LOx structure when submitted to increased substrate concentrations. The results found by fluorescence spectroscopy and dynamic light scattering of LOx solutions, evidenced conformational changes of the enzyme, occurring in presence of inhibitory substrate concentrations. Therefore, the inhibition behavior found at the biosensor, is an outcome of LOx structural alterations as result of a pH-dependent mechanism promoted at high substrate concentrations.Spanish Ministry of Science and Innovation (MICINN), Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (FEDER) (TEC20013-40561-P and MUSSEL RTC-2015-4077-2). Hugo Cunha-Silva would like to acknowledge funding from the Spanish Ministry of Economy (BES-2014-068214

Voltammetric aptasensors for protein disease biomarkers detection: a review

"Available online 24 May 2016"An electrochemical aptasensor is a compact analytical device where the bioreceptor (aptamer) is coupled to a transducer surface to convert a biological interaction into a measurable signal (current) that can be easily processed, recorded and displayed. Since the discovery of the Systematic Evolution of Ligands by Enrichment (SELEX) methodology, the selection of aptamers and their application as bioreceptors has become a promising tool in the design of electrochemical aptasensors. Aptamers present several advantages that highlight their usefulness as bioreceptors such as chemical stability, cost effectiveness and ease of modification towards detection and immobilization at different transducer surfaces. In this review, a special emphasis is given to the potential use of electrochemical aptasensors for the detection of protein disease biomarkers using voltammetry techniques. Methods for the immobilization of aptamers onto electrode surfaces are discussed, as well as different electrochemical strategies that can be used for the design of aptasensors.The authors acknowledge the financial support from the Strategic funding of UID/BIO/04469/2013 unit, from Project POCI-01-0145- FEDER-006984 – Associate Laboratory LSRE-LCM funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT - Fundação para a Ciência e a Tecnologia and project ref. RECI/BBB-EBI/ 0179/2012 (project number FCOMP-01-0124-FEDER-027462) and S. Meirinhos's doctoral grant (ref SFRH/BD/65021/2009) funded by Fundação para a Ciência e a Tecnologia

Electrochemical lactate biosensor based upon chitosan/carbon nanotubes modified screen-printed graphite electrodes for the determination of lactate in embryonic cell cultures.

l-lactate is an essential metabolite present in embryonic cell culture. Changes of this important metabolite during the growth of human embryo reflect the quality and viability of the embryo. In this study, we report a sensitive, stable, and easily manufactured electrochemical biosensor for the detection of lactate within embryonic cell cultures media. Screen-printed disposable electrodes are used as electrochemical sensing platforms for the miniaturization of the lactate biosensor. Chitosan/multi walled carbon nanotubes composite have been employed for the enzymatic immobilization of the lactate oxidase enzyme. This novel electrochemical lactate biosensor analytical efficacy is explored towards the sensing of lactate in model (buffer) solutions and is found to exhibit a linear response towards lactate over the concentration range of 30.4 and 243.9µM in phosphate buffer solution, with a corresponding limit of detection (based on 3-sigma) of 22.6µM and exhibits a sensitivity of 3417±131µAM(-1) according to the reproducibility study. These novel electrochemical lactate biosensors exhibit a high reproducibility, with a relative standard deviation of less than 3.8% and an enzymatic response over 82% after 5 months stored at 4°C. Furthermore, high performance liquid chromatography technique has been utilized to independently validate the electrochemical lactate biosensor for the determination of lactate in a commercial embryonic cell culture medium providing excellent agreement between the two analytical protocols