Hybridization Chain Reaction-Amplified Electrochemical DNA-Based Sensors Enable Calibration-Free Measurements of Nucleic Acids Directly in Whole Blood

Hybridization chain reaction (HCR) amplification strategy has been extensively explored for the application of electrochemical DNA-based sensors. Despite the enhancement in its sensitivity using the HCR, such sensor platform exhibited significant sensor-to-sensor variations in current due to variations in probe counts and lengths. To circumvent this, we are developing here a calibration-free "O-N" approach to generate a ratiometric, unitless value that is independent of these variations. Specifically, this approach employs two types of redox reporters, denoted as "One reporter" and "N reporters", with the former attached on the capture DNA and the latter on H1 and H2 strands. By optimizing the attachment sites of these reporters onto DNA strands, we demonstrate a significantly enhanced sensitivity of such sensor platform by four orders of magnitude, achieving accurate, calibration-free measurement of nucleic acids including ctDNA directly in undiluted whole blood without the requirement to calibrate each individual sensor.