Ultrasensitive Single-Molecule Enzyme Detection and Analysis Using a Polymer Microarray

This report describes a novel method for isolating and detecting individual enzyme molecules using polymer arrays of picoliter microwells. A fluidic flow-cell device containing an array of microwells is fabricated in cyclic olefin polymer (COP). The use of COP microwell arrays simplifies experiments by eliminating extensive device preparation and surface functionalization that are common in other microwell array formats. Using a simple and robust loading method to introduce the reaction solution, individual enzyme molecules are trapped in picoliter microwells and subsequently isolated and sealed by fluorinated oil. The sealing is stable for hours in the COP device. The picoliter microwell device can measure enzyme concentrations in the low-femtomolar range by counting the number of active wells using a digital read-out. These picoliter microwell arrays can also easily be regenerated and reused