Amperometric Detection of Superoxide Dismutase at Cytochrome c-Immobilized Electrodes: Xanthine Oxidase and Ascorbate Oxidase Incorporated Biopolymer Membrane for in-vivo Analysis

Abstract

Superoxide radical is highly reactive and short-lived and is formed in all aerobic cells. The superoxide radical generated during the "respiratory burst" of phagocytic cells plays an important role in the destruction of microorganisms invading into our body. But it may be involved in oxidative damage to tissues and organisms if it exceeds the level at which the systems are able to provide defence. The ubiquitous presence of SOD enzyme throughout the evolutionary chain emphasizes the importance of superoxide radical in cell function and survival. SOD plays a major protective role in living cells and has been widely used as a pharmacological tool in the study of pathophysiological mechanisms. 1-6 Detection of superoxide and SOD has been intensively pursued by spectrophotometric and electrochemical methods. [7][8][9][10][11][12][13][14][15][16] Electrochemical sensors are of great interest due to their advantages, which include microfabrication and in-vivo and in-vitro applications. 17,18 The hemoprotein, cytochrome c (cyt c), undergoes facile reductive reaction with superoxide. Electrochemical biosensors for superoxide with cyt c as the sensing element were constructed employing a suitable electron-transfer promoter.11-14 SOD is a selective scavenger of superoxide, and the best way for the detection of SOD thus could be the estimation of superoxide. Superoxide undergoes spontaneous disproportionation to produce H2O2, an electroactive species. Thus, the electrical response of the superoxide sensor system for interfering compounds is a very important limitation in its application. We recently reported a selective detection of superoxide and SOD at cyt c/alkanethiol electrodes, where the response due to interferents was suppressed by the use of a mixed-monolayer of alkanethiols for anchoring cyt c; but the presence of L-ascorbic acid, a potential scavenger of superoxide existing ubiquitously in our body, was detrimental to the detection of superoxide and SOD. 19 The detection of superoxide and consequently SOD in the presence of L-ascorbic acid was established using XOD/cyt c-bilayer electrodes, where the poly-L-lysine: polystyrenesulfonate complex membrane employed for the immobilization of XOD restricted the permeation of L-ascorbic acid across the membrane and consequently suppressed the scavenging of the superoxide produced at the membrane-bound XOD by Lascorbic acid. 20 The XOD/cyt c-bilayer electrodes could detect SOD in the presence of L-ascorbic acid; however, the sensitivity of XOD/cyt c-bilayer electrodes for SOD was rather low (detection limit, 0.2 µg ml -1 ) relative to that of cyt c-monolayer electrodes (detection limit, 3 ng ml -1 ); 19,20 the permeation of SOD across the polyion complex membrane confining XOD was suppressed in the case of the bilayer electrodes, which resulted into a low sensitivity for the detection of SOD. In the present investigation, the cyt c-immobilized monolayer electrodes were prepared by covalently-immobilizing cyt c on mixed-monolayers of 3-mercaptopropionic acid (MPA) with t...

A new, versatile field immunosensor for environmental pollutantsDevelopment and proof of principle with TNT, diuron, and atrazine

“…First experiments with covalent immobilization of coating protein (Protein A/G) through self-organized monolayers (SAMs) of thioalkanes (method adapted according to Li et al, 1996;Gobi and Mizutani, 2001) eventually allowed binding and inhibition, but we could not reproduce it, probably because of the instability of SAMs in time (data not shown). However, this was an approach which will be taken further because the covalently immobilized Protein A/G could be easily regenerated, and would be therefore a versatile surface for antibody binding and regeneration.…”

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

“…Binding of the biomarker by the recognition molecule results in a local change of potential or current that can be detected by simple measurement. To date the recognition moiety can range from enzymes, [4][5][6][7][8] antibodies, [9][10][11][12] whole cells, [13][14][15][16] receptors, [17][18][19][20] peptides [21][22][23] and DNA 24 and have largely focused on larger, biological molecules. [25][26][27][28] Research efforts in this arena have concentrated mainly on immunosensors where an immunoglobulin is immobilized with a redox active core (such as ferrocene) and used to detect an antibody of interest.…”