Simplified Nitrate-Reductase-Based Nitrate Detection by a Hybrid Thin-Layer Controlled Potential Coulometry/Spectroscopy Technique

Abstract

A novel method for the detection of nitrate was developed using simplified nitrate reductase (SNaR) that was produced by genetic recombination techniques. The SNaR consists of the fragments of the Mo-molybdopterin (MO-MPT) binding site and nitrate reduction active site and has high activity for nitrate reduction. The method is based on a unique combination of the enzyme-catalyzed reduction of nitrate to nitrite by thin-layer coulometry followed by spectroscopic measurement of the colored product generated from the reaction of nitrite with Griess reagents. Coulometric reduction of nitrate to nitrite used methyl viologen (MV(2+)) as the electron transfer mediator for SNaR and controlled potential coulometry in an indium tin oxide (ITO) thin-layer electrochemical cell. Absorbance at 540 nm was proportional to the concentration of nitrate in the sample with a linear range of 1-160 μM and a sensitivity of 8000 AU M(-1). The method required less than 60 μL of sample. Detection of nitrate could also be performed by measuring the charge associated with coulometry. However, the spectroscopic procedure gave superior performance because of interference from the large background charge associated with coulometry. Results for the determination of nitrate concentration in several natural water samples using this device with spectroscopic detection are in good agreement with analysis done with a standard method.

Optically Transparent Carbon Nanotube Film Electrode for Thin Layer Spectroelectrochemistry

“…The R s obtained from the Nyquist plot was in agreement with the measured resistances in the scan rate study: 1871 ohm (1L), 997 ohm (2L), 543 ohm (3L), 403 ohm (4L), 297 ohm (5L). However, little kinetic information can be extracted because the 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 system was dominated by the high R s , indicated by a lack of R ct in the Nyquist plot ( Figure 7A) caused by very fast heterogeneous electron transfer for the Fe(CN) 6 3-/Fe(CN) 6 4-redox couple. In order to study the intrinsic charge transfer resistance, CNT films were then applied on a conductive GC electrode, as described above, and Pt wire was used as the reference electrode to further reduce the system resistance.…”

“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Pt wire was used as the reference electrode. 5 mM of equal concentrations of K 3 Fe(CN) 6 /K 4 Fe(CN) 6 in 1 M KCl was the analyte and supporting electrolyte. Inset: zoom in of the higher frequency range of the impedance.…”

“…1 Currently, transparent electrode materials such as grids or thin films of Au, Pt, indium tin oxide (ITO), and carbon and fiber optics that allow the transmission of the changes occurring at or adjacent to the electrode surface are commonly used. [3][4][5][6] A narrow thickness of the electrolyte layer contacting the working electrode shortens the time for complete electrolysis in the optically transparent thin layer electrode (OTTLE). [5][6][7] The spectroelectrochemical properties of the analytes are then monitored by controlling the applied potential.…”

“…[3][4][5][6] A narrow thickness of the electrolyte layer contacting the working electrode shortens the time for complete electrolysis in the optically transparent thin layer electrode (OTTLE). [5][6][7] The spectroelectrochemical properties of the analytes are then monitored by controlling the applied potential. 1 Spectroelectrochemistry in the UV and IR regions is often limited by the absorption of electrolyte and electrode material itself.…”

“…Using this setup, the R s of layers of CNT films was reduced to a few ohms as shown in Figure 7B. The R ct of Fe(CN) 6 3− /Fe(CN) 6 4− redox couple at the electrode interface was detected at this CNT modified GC electrode. Starting from 2 layers of CNT films, stretched out semicircles were obtained, which have typically been observed for porous structures of CNT film.…”