Diazonium modified gold microelectrodes onto polyimide substrates for impedimetric cytokine detection with an integrated Ag/AgCl reference electrode

This paper describes a simple technique for the fabrication of a gold microelectrode array with an integrated Ag/AgCl reference microelectrode based on polyimide (PI) which is a flexible and biocompatible thermoplastic substrate. The investigated fabrication technology was based on soft lithography which involved a combination of replica molding (RM) and patterning of self-assembled monolayers (SAMs), through microcontact printing (μCP). From a fabricated silicon microelectrode mold, an elastomeric stamp of the microelectrodes was replicated in polydimethylsiloxane (PDMS) and inked with octadecanethiol (ODT) for patterning of the gold surface. The non-patterned gold was then removed by selective chemical etching leaving very well defined gold microelectrodes with a high resolution. Finally, the integrated Ag/AgCl reference microelectrode was fabricated by silver electro-deposition onto the gold microelectrode. The developed biosensor was then tested for a medical application by immobilizing both monoclonal antibodies (mAb); anti-human interleukin-10 (IL-10) and anti-human tumor necrosis factor-alpha (TNF-α) onto different working electrodes. Functionalization of the gold microelectrodes was made with carboxyl diazonium through cyclic voltammetry (CV). The multiple detection of the corresponding cytokines was verified by fluorescence, whilst electrochemical impedance spectroscopy (EIS) analysis quantified a detectable IL-10 cytokine concentration from 1 to 15 pg/mL. This confirmed the biorecognition between the anti-human mAb (IL-10 and TNF-α) and of its corresponding cytokine. The obtained results show that the mAbʹs were efficiently immobilized and the proteins for human IL-10 were detectable at weak concentrations. The electrochemical measurements were applied on the manufactured gold microelectrodes for ∼9 h. This shows a high resistance of the microelectrodes for long-lasting measurements and, therefore, highlights the good adhesion of gold-PI. The results presented here have demonstrated the feasibility of a simple microelectrode fabrication technology with application to a biomedical response.