NADH detection interference

Agboola et al. [57] reported the utilization of phenylamine-modified SWCNTs integrated with cobalt(II) octa[(3,5-biscarboxylate)-phenoxy] phthalocyanine (CoOBPPc) for (5-nicotinamide adenine dinucleotide (NADH) detection (Fig. 10a). The sensor showed good ability to detect low concentrations of NADH even in the presence of high concentration of ascorbic acid, which frequently interferes in the electrochemical determination of this analyte. Comparative cyclic voltammograms at bare GC and CNTs-modified electrode can be seen in Fig. 10b. 

The problem associated with amperometric detection of NADH is similar to that of H202, with a very high overpotential required [3,4] and with electrode fouling due to the presence of radical intermediates produced during NADH oxidation that then interfere with the measurement [5]. 

Coupled reactions of two or more enzymes can also be used to minimize interference, as well as to amplify the response and extend the scope of the enzyme electrode towards additional analytes. For example, peroxidases can be coupled with oxidases to allow low-potential detection of the liberated peroxide. Electrocatalytic surfaces, particularly those based on metallized carbon, represent a new and effective approach for minimizing electroactive interference [9]. Such strategy relies on the preferential electrocatalytic detection of the liberated peroxide or NADH species at rhodium or ruthenium dispersed carbon bioelectrodes. 

The precolumn was first characterized by evaluating the retention times of NADH and the known electrochemical interferences. Spectroscopic detection was used for this, since the electrode passivators (IgG, HSA), uric acid, and NADH absorb at 280 nm, and IgG and HSA are electroinactive at 750 mV versus Ag/AgCI. Figure 16 shows a chromatogram of a blank human serum—NADH mixture which was injected into the Lichrosorb-DIOL precolumn. The first peak (retention time of 113 sec) was primarily composed of IgG and HSA. The second peak (173 sec) was NADH the shoulder that appears at approximately 190 sec was uric acid. Therefore a heart-cut should be from 150 sec (to remove the macro-molecular fraction) to 185 sec (to remove the late-eluting uric acid among other possible interferences). 

Milczarek, G. and A. Ciszewski (2001). Voltammetric detection of NADH free from interference of ascorbic acid using a glassy carbon electrode modified with an elec-tropolymerised porphyrin film. Electroanalysis 13(2), 164—166. 

Excess NADH is then oxidized to NAD to eliminate interference with color development when NO2 from Reaction 18-9 is measured colorimetrically by reactions such as 18-7. For quantitative analysis in the field, a small, battery-operated spectrophotometer can be used with a set of nitrate standards. Alternatively, color can be compared visually with a chart showing colors from several standards. Commercial field kits allow analysis in the range 0.05-10 ppm nitrate nitrogen. Laboratory apparatus provides a precision of 2% when measuring 0.2 ppm NO3 nitrogen, with a detection limit of 3 ppb. Nitrate reductase has been applied to the measurement of nitrate in a classroom aquarium. ... 

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