A publication in the journal Micromachines describes how researchers managed to control the concentrations of DNA-binding dyes in single-molecule optical tweezers experiments on DNA. They established a controlled concentration gradient with the multi-channel laminar microfluidics system integrated with their C-Trap® correlated optical tweezers and confocal microscopy.
The study demonstrates a new approach to study concentration-dependent mechanical properties on the same biomolecule, without the need to change the sample or its surrounding solution.
The researchers first established a concentration gradient of DNA intercalating dyes, including SYTOX Orange (SxO), through two adjacent microfluidic channels (containing a buffer or dye, respectively) at a constant flow. Next, they moved a DNA molecule, tethered between two optically trapped beads, along the concentration gradient at predefined speeds.
With this setup, Kretzer et al. characterized the concentration gradient in two ways. They monitored the fluorescence intensity of SxO or, since intercalating dyes like SxO lengthens the DNA contour length, they also plotted changes in DNA lengths as a function of the gradient.
With the integrated confocal microscopy, the researchers could correlate the fluorescence intensity of the intercalating SxO and the contour length of the DNA molecule. In other words, moving the captured molecule to higher SxO concentrations relaxed the DNA – a process that can be reversed upon moving the DNA down the gradient.
The results confirmed that the combination of optical tweezers, fluorescence microscopy, and microfluidics-controlled ligand gradients, serve to assess concentration-dependent functions of ligands on biomolecule dynamics.
“The major advantage of the use of the concentration gradient is that uncertainties of ligand concentration adjustment caused by the adsorption and desorption of cyanine dyes to and from the surfaces of the microfluidic device can be alleviated,” the authors concluded.
For more information, read the full article published in Micromachines and entitled “Single-Molecule Mechanics in Ligand Concentration Gradient”.