Label-free electrochemical immunosensor based on K3[Fe(CN)6] as signal for facile and sensitive determination of tumor necrosis factor-alpha

We herein proposed a novel, facile, and sensitive strategy for selective detection of tumor necrosis factor-alpha (TNF-α) using K3[Fe(CN)6] as the label-free signal. A K3[Fe(CN)6]-chitosan-glutaraldehyde system (K-CS-GA) capable of high signal indication was developed for the first time to improve the sensitivity of the electrochemical immunosensor. K-CS-GA acting as the signal indicator was first fixed on a glass carbon electrode to be the signal layer. Subsequently, nafion was used to cover the signal layer and immobilize mouse anti-human TNF-α onto the electrode. The target TNF-α captured onto the electrode through immunoreactions efficiently induced the current response of the electrode due to the non-conducting biomoleculars. TNF-α was detected by monitoring the electrode current response in PBS using cyclic voltammetry (CV), which proved to be sensitive to varying concentrations of TNF-α. The decreases of anodic currents were proportional to the logarithm of the antigen concentrations in the range of 0.02–34 ng/ml with the detection limit of 10 pg/ml. The proposed immunosensor not only demonstrated high sensitivity, good selectivity, acceptable stability, and reproducibility, but also exhibited potential applications in clinical protein diagnostics.