Real-Time Impedimetric Detection of DNA Amplification By PCR Using Electrochemical Impedance Spectroscopy

DNA sensors are innovative diagnostic tools that have gained broad acceptance in recent years. Different procedures have been used to develop these sensors. One of the most frequently used method is the self-assemble monolayer (SAMs) that provides advantages in terms of simplicity, efficiency and cost of electrode design. Gold surface modification with SAMs change the electrochemical capacitive behavior of their surface which can be measured by Electrochemical Impedance Spectroscopy (EIS). Tethering of ssDNA via SAMs techniques onto metallic Au surface provide a biosensing probe which is sensitive to changes on the surface, and these changes on the ssDNA conformation correlate to changes in the double layer capacitance (C_dl). Using this as the basis, a novel microchip with an interdigitated array electrodes was done and used as a real-time electrochemical biosensor for the detection of DNA amplification directly on its surface. The microchip was designed using a silicon/SiO₂ substrate and gold thin film patterned by photolithography. The microchip is connected by a USB type A connector. In this device, the polymerase chain reaction (PCR) takes place on the surface interface, and the primer ssDNA tethered to the surface is elongated. Every modification, variation or alteration of the DNA on the surface of the electrodes result in changes in the C_dl and measured by EIS having a non-faradaic and label-free detection. The recognized sensitivity of electrochemical platforms and the well-known specificity of exclusive primers provide portability, reliability, rapidness, and accuracy in a robust biosensor microchip. Moreover, by avoiding the use of optical labels we are able to measure complex or unpurified biofluid samples without the need of extensive purification. All these features will have a tremendous impact on the biomedical science filling the absence of methods that can detect DNA in real-time and in a direct readout of crude biological fluids.