Abstract This proposal is in response to NIH FOA (PAR-22-126), which “supports exploratory research leading to the development of innovative technologies for biomedical research...” This R21 project aims to harness the high sensitivity and resolving power of optical microscopy and asymmetric bipolar microelectrode arrays to develop a low-cost, highly sensitive bipolar electrochemical array platform for detecting and counting individual biological target species. Many biologically or clinically relevant species, such as certain cancer biomarkers, present at very low concentrations, sometimes down to a few copies. These species have been difficult to detect and quantify with existing bioanalytical methods due to their insufficient sensitivity, selectivity, and response speed. To address this challenge, we propose to develop a bipolar electrochemical single entity bioanalyzer, which will allow us to analyze individual biological species, such as single viruses and circulating tumor exosomes. The success of this project builds upon the strong expertise of the PI’s group in bipolar electrochemical arrays, microfabricated sensors, and single entity electroanalysis and has three specific aims. Aim 1 builds the asymmetric bipolar microelectrode arrays containing 250,000 electrodes and characterizes and optimizes them for single entity analysis. Aim 2 will synthesize and characterize Janus Pt/silica nanoparticles (NPs) and use them to label analyte species pre-concentrated onto magnetic microbeads. Aim 3 will build an integrated analytical platform by combining the bipolar microelectrode array with optical microscopy, characterize and optimize its analytical performance for single entity bioanalysis of pseudovirus particles and exosomes. The proposed analytical platform is innovative and powerful for single entity bioanalysis due to the use of an optical signal to amplify and read a small electrochemical signal. The use of a large array of 250,000 bipolar microelectrodes and magnetic beads for pre-concentration further enhances the sensitivity, selectivity, and throughput. Future work will develop a standalone benchtop analytical instrument, which will be useful for their general use in research and diagnosis involving single entity bioanalysis.