Detection of the superoxide radical anion using various alkanethiol monolayers and immobilized cytochrome c.
The superoxide radical anion (SO) is a critical biomarker for monitoring cellular stress responses. Electrochemical SO biosensors are frequently constructed through the covalent immobilization of cytochrome c (Cyt c) onto self-assembled monolayers (SAMs); however, a detailed comparison of these systems as well as configuration influence on SO detection is needed to enable robust applications. Two reaction pathways, oxidation of SO by the SAM-modified gold electrode or electron transfer through a protein and monolayer relay, may be involved during the electrochemical detection of SO with Cyt c, depending on the SAM that is used. Although electrodes with SAMs alone can exhibit a high sensitivity and low limit of detection (LOD) for the SO, they can suffer from a strong response to the presence of interferents such as hydrogen peroxide and ascorbic acid. Electrodes with immobilized Cyt c show decreased sensitivity, but exhibit better selectivity and resistance to fouling in complex media. Considering the trade-offs between sensitivity, selectivity, and LOD for SO detection, a bioelectrode made with Cyt c immobilized on dithiobis(succinimidyl)propionate (DTSP) appears to be the most suitable configuration. In phosphate buffer, the DTSP/Cyt c electrode has a sensitivity of 410 nA microM(-1) cm(-2) and an LOD for SO of 73 nM. Results are also presented for the detection of SO in a complex tissue culture media (MEM) with and without serum, and the sensitivity of the DTSP/Cyt c in MEM in the absence of serum increased to 640 nA microM(-1) cm(-2). By measuring SO with a DTSP/Cyt c electrode before and after the addition of a bolus of the superoxide dismutase (SOD) enzyme, the specificity of the SOD enzyme can be combined with the sensitivity of Cyt c system.
Chen
Xiaojun J
XJ
Department of Chemical Engineering, Columbia University, New York, New York 10027, USA.
West
Alan C
AC
Cropek
Donald M
DM
Banta
Scott
S
D000818:Animals; D015374:Biosensing Techniques; D002384:Catalysis; D002417:Cattle; D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry*; D055617:Micro-Electrical-Mechanical Systems; D013388:Succinimides / Q000737:chemistry; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000032:analysis*; D019820:Xanthine / Q000737:chemistry; D014969:Xanthine Oxidase / Q000737:chemistry
D004800:Enzymes, Immobilized; D013388:Succinimides; D013481:Superoxides; D019820:Xanthine; D045304:Cytochromes c; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; C011240:dithiobis(succinimidylpropionate)