Strategies for the development of an electrochemical bioassay for TNF-alpha detection by using a non-immunoglobulin bioreceptor

TNF-α is an inflammatory cytokine produced by the immune system. Serum TNF-α level is elevated in some pathological states such as septic shock, graft rejection, HIV infection, neurodegenerative diseases, rheumatoid arthritis and cancer. Detecting trace amount of TNF-α is, also, very important for the understanding of tumor biological processes. Detection of this key biomarker is commonly achieved by use of ELISA or cytofluorimetric based methods. In this study the traditional optical detection was replaced by differential pulse voltammetry (DPV) and an affinity molecule, produced by evolutionary approaches, has been tested as capture bioreceptor. This molecule, namely a combinatorial non-immunoglobulin protein (Affibody®) interacts with TNF-α selectively and was here tested in a sandwich assay format. Moreover magnetic beads were used as support for bioreceptor immobilization and screen printed carbon electrodes were used as transducers. TNF-α calibration curve was performed, obtaining the detection limit of 38 pg/mL, the quantification range of 76-5000 pg/mL and RSD%=7. Preliminary results of serum samples analysis were also reported.