Sensitive single‐molecule protein quantification and protein complex detection in a microarray format

Single‐molecule protein analysis provides sensitive protein quantitation with a digital read‐out and is promising for studying biological systems and detecting biomarkers clinically. However, current single‐molecule platforms rely on the quantification of one protein at a time. Conventional antibody microarrays are scalable to detect many proteins simultaneously, but they rely on less sensitive and less quantitative quantification by the ensemble averaging of fluorescent molecules. Here, we demonstrate a single‐molecule protein assay in a microarray format enabled by an ultra‐low background surface and single‐molecule imaging. The digital read‐out provides a highly sensitive, low femtomolar limit of detection and four orders of magnitude of dynamic range through the use of hybrid digital‐analog quantification. From crude cell lysate, we measured levels of p53 and MDM2 in parallel, proving the concept of a digital antibody microarray for use in proteomic profiling. We also applied the single‐molecule microarray to detect the p53–MDM2 protein complex in cell lysate. Our study is promising for development and application of single‐molecule protein methods because it represents a technological bridge between single‐plex and highly multiplex studies.