Development of an Amperometric Biosensor Platform for the Combined Determination of l-Malic, Fumaric, and l-Aspartic Acid

Abstract

Three amperometric biosensors have been developed for the detection of L-malic acid, fumaric acid, and L -aspartic acid, all based on the combination of a malate-specific dehydrogenase (MDH, EC 1.1.1.37) and diaphorase (DIA, EC 1.8.1.4). The stepwise expansion of the malate platform with the enzymes fumarate hydratase (FH, EC 4.2.1.2) and aspartate ammonia-lyase (ASPA, EC 4.3.1.1) resulted in multi-enzyme reaction cascades and, thus, augmentation of the substrate spectrum of the sensors. Electrochemical measurements were carried out in presence of the cofactor β-nicotinamide adenine dinucleotide (NAD) and the redox mediator hexacyanoferrate (III) (HCFIII). The amperometric detection is mediated by oxidation of hexacyanoferrate (II) (HCFII) at an applied potential of + 0.3 V vs. Ag/AgCl. For each biosensor, optimum working conditions were defined by adjustment of cofactor concentrations, buffer pH, and immobilization procedure. Under these improved conditions, amperometric responses were linear up to 3.0 mM for L-malate and fumarate, respectively, with a corresponding sensitivity of 0.7 μA mM (L-malate biosensor) and 0.4 μA mM (fumarate biosensor). The L-aspartate detection system displayed a linear range of 1.0-10.0 mM with a sensitivity of 0.09 μA mM. The sensor characteristics suggest that the developed platform provides a promising method for the detection and differentiation of the three substrates.

Application of a Portable Multi-Analyte Biosensor for Organic Acid Determination in Silage

“…In comparison to conventional analytical methods, the described biosensor system offers the advantages of rapid and simultaneous detection, and capability for repeated application due to long-term stability. For particular requirements, the flexible system can be easily enhanced with additional electrodes for further analytes [ 23 ]. Future investigations will focus on application of the biosensor in different biological samples and evaluation of the compact and portable measurement device for long-term and on-site monitoring of fermentation processes.…”

“…We have recently reported on the development and extensive optimization of different enzyme-based biosensor arrays for the parallel determination of several analytes [ 22 , 23 ]. The combination of different dehydrogenases and a diaphorase, required for the amperometric detection principle and regeneration of the cofactor nicotinamide adenine dinucleotide (NAD) [ 24 , 25 ], allowed construction of electrochemical multi-parametric biosensors.…”

Toward a Hybrid Biosensor System for Analysis of Organic and Volatile Fatty Acids in Fermentation Processes

“…Purification of SCAOx was accomplished by affinity chromatography with a Strep-Tactin Macroprep column (IBA GmbH, Göttingen, Germany) as outlined earlier (Röhlen et al, 2017 ). Briefly, cell pellets were suspended in 100 mM sodium phosphate buffer (pH 7.5, supplemented with 150 mM NaCl and 10 μM FAD) and lysed by sonication.…”

Challenges in Using Precision Agriculture to Optimize Symbiotic Nitrogen Fixation in Legumes: Progress, Limitations, and Future Improvements Needed in Diagnostic Testing

“…However, this method has limitations for use under field conditions in the short-medium term, as the GlnLux method cannot distinguish N derived from SNF versus soil-uptake N. This method has potential for precision agriculture if translated into a field-based technology, by conversion into an amperometric biosensor specific for gln [72] that can be linked to variable rate fertilization. There is also potential to engineer additional amperometric biosensors that can detect other fixed-N metabolites [73], as well as inorganic N [74]. Chemical-based tests are currently available (e.g., Merkoquant test strips, Reflectoquant strips) to measure plant nitrate concentration from plant sap at the field level [39].…”