Abstract
A SPEC/AuNPs/PMB modified electrode was prepared by electrodeposition and electro-polymerization. The electrochemical behavior of reduced nicotinamide adenine dinucleotide (NADH) on the surface of the modified electrode was studied by cyclic voltammetry. A certain amount of substrate and glutamate dehydrogenase (GLDH) were coated on the modified electrode to form a functional enzyme membrane. The ammonia nitrogen in the water sample could be calculated indirectly by measuring the consumption of NADH in the reaction. The results showed that the strength of electro-catalytic current signal was increased by two times; the catalytic oxidation potential was shifted to the left by 0.5 V, and the anti-interference ability of the sensor was enhanced. The optimum substrate concentration and enzyme loading were determined as 1.3 mM NADH, 28 mM α-Ketoglutarate and 2.0 U GLDH, respectively. The homemade ceramic heating plate controlled the working electrode to work at 37 °C. A pH compensation algorithm based on piecewise linear interpolation could reduce the measurement error to less than 3.29 μM. The biosensor exhibited good linearity in the range of 0~300 μM with a detection limit of 0.65 μM NH4+. Compared with standard Nessler’s method, the recoveries were 93.71~105.92%. The biosensor was found to be stable for at least 14 days when refrigerated and sealed at 4 °C.
Highlights
Glutamate dehydrogenase (GLDH), alanine dehydrogenase (AlaDH), leucine dehydrogenase (LeuDH), phenylalanine dehydrogenase (PheDH) and valine dehydrogenase (ValDH) are the main members of the amino acid dehydrogenase family, all of which are NAD(P)+ dependent [23,24], and their enzymatic reactions are accompanied by catalytic reduction ammoniation
During the first 10 cycles of scanning, the peak current increased with the increase of scanning times, indicating that a layer of polymethylene blue film was formed on the surface of the working electrode
By analyzing the measurement principle of the biosensor, it can be found that each measurement of the biosensor is accompanied by the consumption of mixed substrate (NADH, PMB, α-Ketoglutarate) and the loss of enzyme (GLDH) activity, which are the main reasons for the poor reusability of the electrode
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The detection methods of ammonia nitrogen in aquaculture water mainly include spectrophotometry and colorimetry [3]. The ammonia gas sensing electrode method has the advantages of high precision, strong anti-interference and fast response, which is suitable for automatic and continuous determination of ammonia nitrogen [10,11]. This method requires the pH value of the solution to be greater than 11. The researchers indirectly predicted the concentration of ammonia nitrogen by measuring the consumption of coenzyme NADH in enzymatic reaction. The thermostatic controller and pH compensation method were designed to further improve the measurement accuracy of the sensor
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