Label-Free Multiplexed Electrical Detection of Cancer Markers on a Microchip Featuring an Integrated Fluidic Diode Nanopore Array

We introduce an integrated array of glass nanopores on a silicon microchip fabricated in a batch process through low-resolution photolithography and standard semiconductor processing tools. By functionalizing each nanopore against a distinct target, we further demonstrate ultrasensitive, label-free, multiplexed electrical detection of cancer-marker proteins in real time through charge-dependent ionic current rectification. As nanofluidic diode biosensors, the nanopores return rapid results, with a limit of detection reaching concentrations as low as attomolars in assay buffer and femtomolars in undiluted untreated human serum, a rare achievement for this class of nanosensors. Multiplexed detection capability has been demonstrated on proteins carcinoembryonic antigen, alpha-fetoprotein antigen, and human epidermal growth factor receptor-2, with the assay further scalable to a size that is limited by the readout electronics. The nanopores are also found with a considerably advanced detection limit as well as dynamic range in relation to the nanoslit counterparts, validated by the measurements on cardiac protein troponin T. This highly robust assay platform draws from rich nanopore physics and could provide further enhanced detection through concentration polarization, subsequent target enrichment, and serum desalting, all potentially induced by the nanopores presently redundant in the array. This integration would be crucial for removing major obstacles for the practical use of nanopore-based assays.