Rapid Microfluidic Double-Jump Mixing Device for Single-Molecule Spectroscopy

Abstract

We introduce a microfluidic double-jump mixing device for investigating rapid biomolecular kinetics with confocal single-molecule spectroscopy. This device enables nonequilibrium dynamics to be probed, e.g., transiently populated intermediates that are inaccessible with existing single-molecule approaches. We demonstrate the potential and reliability of the method on time scales from milliseconds to minutes by investigating the coupled folding and binding reaction of two intrinsically disordered proteins and the conformational changes occurring in a large cytolytic pore-forming toxin.

High-throughput, non-equilibrium studies of single biomolecules using glass-made nanofluidic devices

“…Additionally, we observed polymerization of 25 bases on a DNA template by a DNA polymerase, illustrating that complex biological reactions can be followed in real time and in a continuous fashion. In our current mixing design, the distance from junction to outlet is 100 μm, corresponding to a maximum residence time in the nanochannel of around 10 s. To gain access to further time points after mixing, designs using meandering channels could be implemented as demonstrated for confocal microscopy 37,38 or widefield microscopy. 39 Furthermore, our current field of view is cropped by a factor of two do ensure data acquisition at 100 Hz.…”

Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

“…Mixing devices have been primarily demonstrated for confocal applications, with latest designs even enabling two consecutive mixing steps 11,30 . Here, for the first time, we use the PFC principle for a mixing geometry: Two syringe pumps deliver flow to two microchannels and two respective parallel feeding nanochannel inlets which merge to a single nanochannel at a T-junction (Fig.…”

“…To gain access to further time points after mixing, designs using meandering channels could be implemented as demonstrated for confocal microscopy 11,30 or widefield microscopy. 36 Furthermore, our current field of view is cropped by a factor of two do ensure data acquisition at 100 Hz.…”

Fluorescence Redox Blinking Adaptable to Structural Analysis of Nucleic Acids

“…[24][25][26][27][28][29][30] In addition, single-molecule measurements can also be performed in combination with ac onventional method such as stopped flow. [31] We have harnessed the phenomenon known as blinking, which is unique to single-molecule fluorescencem easurements, to investigate structural switching. In single-molecule measurements, fluorescencef luctuationsa re observed under continuous-wave laser illumination, and this fluctuating behavior is knowna sb linking.…”

“…Various fluorescence‐based single‐molecule studies have contributed to our understanding of the conformational transitions of nucleic acid and protein structures . In addition, single‐molecule measurements can also be performed in combination with a conventional method such as stopped flow …”