Enhanced adhesion of functional layers by controlled electrografting of ethylenediamine on ITO for electrochemical immunoassay in microfluidic channel

Two-electrode (2E) system of the interdigitated electrode array (IDA), which operates neither reference nor counter electrodes, has great potential to miniaturize multiplex immunoassay in a microfluidic chip for point-of-care testing. However, it is necessary to firmly immobilize the mediator layer on IDA made of indium tin oxide (ITO) which is chemically inert. It is important because the mediator determines the electrochemical potential in the 2E system, but the layer is easy to be detached during the washing processes of immunoassay. Here, we controlled the concentration of ethylenediamine (EDA) to generate a permeable and robust film to adhere to mediators on the ITO IDA chip. Electrooxidation of EDA yielded thin oligomeric ethyleneimine (OEI) film and it provided amine groups for immobilizing the mediator, poly(toluidine blue) (pTB), via common conjugation reaction. Despite repeated flows in the microchannel, which are essential for sensitive immunoassay, the pTB/OEI layer was hardly washed and still remained on the ITO IDA. Myoglobin was measured down to ∼ pg/mL level. Therefore, the ITO IDA modified with the OEI film in the 2E system constituted a stable platform that withstands washing steps for sensitive electrochemical detection in the miniaturized immunoassay.