Enzyme molecules in solitary confinement.

Large arrays of homogeneous microwells each defining a femtoliter volume are a versatile platform for monitoring the substrate turnover of many individual enzyme molecules in parallel. The high degree of parallelization enables the analysis of a statistically representative enzyme population. Enclosing individual enzyme molecules in microwells does not require any surface immobilization step and enables the kinetic investigation of enzymes free in solution.

A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase.

The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics.

Single molecule kinetics of horseradish peroxidase exposed in large arrays of femtoliter-sized fused silica chambers.

Large arrays of femtoliter-sized chambers were etched into the surface of fused silica slides to enclose and observe hundreds of single horseradish peroxidase (HRP) molecules in parallel. Individual molecules of HRP oxidize the fluorogenic substrate Amplex Red to fluorescent resorufin in separate chambers, which was monitored by fluorescence microscopy. Photooxidation of Amplex Red and photobleaching of resorufin have previously limited the analysis of HRP in femtoliter arrays.

Maleimide activation of photon upconverting nanoparticles for bioconjugation.

Photon upconverting nanoparticles (UCNPs) have become an important new class of optical labels. Their unique property of emitting visible light after photo-excitation with near-infrared radiation enables biological imaging without background interference or cell damage. Biological applications require UCNPs that are dispersible in water and allow the attachment of biomolecules.