Real-time glucose monitoring system containing enzymatic sensor and enzymatic reference electrodes

Every electrochemical biosensor uses two or three electrode setup, which involves sensing electrode for a specific reaction, metal/salt reference electrode (i.e., Ag/AgCl or Hg/Hg2Cl2) for the control of the potential and, is some cases, counter electrode for the compensation of the current. This setup has significant flaws related to metal/salt reference electrodes: they are bulky and difficult to miniaturize, leak electrolyte to the medium, lose the ability to define the electrochemical potential precisely in time, consequently, have to be updated or replaced. This causes problems when the biosensor cannot be easily replaced (e.g., implanted electronics). Here we present a fully enzymatic real-time glucose monitoring system capable of referencing its own electrochemical potential. Using sensing electrode composed of wired glucose dehydrogenase and enzymatic reference electrode composed of wired laccase we have created a stable and accurate electrode system, which measured fluxes in concentration of glucose in a physiological range (3-8 mM), and demonstrated performance of the designed system in undiluted human serum. In addition, our designed enzymatic reference electrode is universal and may be applied for other biosensors, thus open possibilities for the new generation of implantable devices for healthcare monitoring.