The Electrochemical Nitrite Ion Sensor Using a Neuroglobin Bonded on Conducting Polymer
Authors: Moon Jong-Min , N. G Gurudatt , Shim Yoon-Bo 
Published: February 2020

The neuroglobin (Ngb) presents in a variety of areas in the brain and expresses differently according to its regions of the brain tissue. In previous studies, several functions of Ngb have been proposed as follows; 1) supply O2 to mitochondria, 2) detoxify reactive oxygen or nitrogen species, 3) convert NO2
- to NO at low O2 pressure, and 4) prevent hypoxia-induced apoptosis. However, its functionality in a biological system and electrochemical characteristic is still uncertain. Ngb could perform as a catalyst for the nitrite oxidation, because the ion appears in the electron transfer chain system in vivo [1]. In addition, the nitrite detection is significant because it adversely effects on human health, since nitrite ions easily react with amines to form toxic, carcinogenic nitrosoamines and they change oxyglobin to metglobin in blood vessel which not only interfere the oxygen transport, but also act as NO storage in the biochemical cycle, which results in the state of hypoxia and ischemia [2]. Therefore, the nitrite concentration can be dependable to quantify the nitric oxide and utilized as a biomarker for individual [3]. Thus, we firstly report the electrochemical properties of Ngb and electrocatalytic reaction for the nitrite detection.

The Ngb modified sensor was fabricated by electrodeposition of gold nanoparticles onto the glassy carbon electrode (AuNPs/GCE). Then, conducting polymer film on the AuNPs/GCE was prepared by electropolymerization of 1.0 mM terthiophene bearing benzoic acid (TTBA) in a 0.1 M TBAP/CH2Cl2 solution. The pTTBA/AuNPs/GCE was immersed in a PBS solution containing 10.0 mM EDC/NHS for the activation of carboxylic acid group. Thereafter, the electrode was washed and subsequently incubated in Ngb solution for 12h. The surface characterization of modified sensor probe was further established by CV, XPS, AFM, and QCM.

CVs were recorded for modified electrodes between 0.5 and -0.5 V at a scan rate of 50 mV/s in 0.1 M PBS (pH 7.4). No peak was observed for AuNPs/GCE, and pTTBA/AuNPs/GCE, while the quasi reversible redox peak was observed at -0.07/ 0.01 V when using Ngb/pTTBA/AuNPs/GCE. Thus, the pair of peaks is resulted from the redox process of the electroactive sites of immobilized Ngb. To investigate the kinetics of electron transfer on the Ngb modified electrode, a variation of scan rate was performed. According to the Laviron’s diffusionless model, the apparent electron transfer rate constant (ks) of the Ngb modified electrode was calculated to be 0.57 s-1. For the amperometric detection of NO2
-, experimental parameters such as temperature, pH, and apply potential were optimized. The linear dynamic range was determined to be from 0.3 to 11.0 μM with the detection limit of 0.1 μM. The detection method was validated by detecting the nitrite released from PC 12 and Schwann cells in cellline.

[1] Marinis, E.D.; Marino, M.; et al., IUBMB Life, 2011
, 63, 140-145.

[2] Shiddiky, Md.A.; Shim, Y.-B.; et al., J. Agric. Food Chem., 2009, 57, 4051-4057.

[3] Koh, W.C.A.; Shim, Y.-B.; et al., Anal. Chem., 2009,83, 6177–6183.