Coupling confocal fluorescence detection and recirculating microfluidic control for single particle analysis in discrete nanoliter volumes

The recent proliferation of platforms designed to handle arrays of nano- and picolitre volumes is in response to the need to perform biological assays on discrete entities, such as single cells. However, a critical challenge associated with this trend for in vitro compartmentalization is the need for highly sensitive, yet low-volume detection platforms. In this paper, we coupled confocal fluorescence detection with recirculating microfluidic control to perform single particle DNA assays within five nL chambers. The performance of this low-volume assay was shown to match that of traditional single molecule detection platforms. However, volume requirements per measurement were nearly 3 orders of magnitude less than conventional systems, enabling future integration with lab-on-a-chip systems that require discrete or digitalized sample processing.