An electrochemical impedance biosensor with aptamer-modified pyrolyzed carbon electrode for label-free protein detection

We present an electrochemical impedance biosensor utilizing pyrolyzed carbon film as a working electrode material for aptamer-based thrombin detection. Batch-fabricated, smooth thin film carbon electrodes, which are fabricated by photolithography and photoresist thermal decomposition at high temperatures in inert ambient, are obtained for integrated electrochemical biosensors. To confirm the suitability of pyrolyzed carbon for use in an electrochemical biosensor, physical and electrical properties of carbon film pyrolyzed with a positive photoresist, AZ9260, were studied. Film thickness after pyrolysis was between 19% and 15% relative to the initial photoresist thickness and the cross-section was changed from rectangular to round shape due to the photoresist reflow characteristics. Resistivity of carbon thin film pyrolyzed at 1000 °C was 3 mΩ cm, which is comparable to that of highly boron-doped polysilicon. The pyrolysis temperature of 1000 °C was chosen in order to obtain carbon film with high conductivity for use as a working electrode. Thrombin aptamer was grafted onto the pyrolyzed carbon surface using carbodiimide-mediated chemistry, followed by Triton-X 100 and BSA treatment to reduce non-specific binding of thrombin. Electron-transfer resistance changes due to thrombin binding onto the carbon surface were measured using electrochemical impedance spectroscopy techniques. Thrombin concentrations between 0.5 nM and 500 nM were detected by electrochemical measurement. Pyrolyzed carbon can provide a new approach for miniaturization, integration, and low-cost fabrication in electrochemical biosensors.