Optical Nanopore Blockade Sensors for Multiplexed Detection of Proteins

Enduring challenges for quantitative analysis in nanopore sensing are the detection of low biomarker concentrations in reasonable time frames and the detection of multiple biomarkers in the same sample. Herein we report an optical blockade nanopore sensor strategy that can detect more than a protein at femtomolar concentrations in rapid time (approximately 12 min). This is done using a nanopore array functionalized with an aptamer that can bind to two different but related target proteins. The assay then monitors two different colors of fluorescent particles modified with antibodies specific to the protein of interest, as they block the nanopores using a wide-field microscope. By distinguishing specific and nonspecific blockade events for each nanoparticle based on whether they can be easily pulled out of the nanopores using an electric field, we can simultaneously quantify the concentrations of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) down to femtomolar concentrations.