Analysis of Protein–Protein Interactions by Using Droplet‐Based Microfluidics

Abstract

Every little drop: The KD values of angiogenin (ANG) interactions as shown by FRET analysis of thousands of pL‐sized droplets agree with data from bulk‐fluorescence polarization measurements. Importantly, the use of fluorophores does not affect the activity of ANG or the binding of anti‐ANG antibodies to ANG. Such an experimental platform could be applied to the high‐throughput analysis of protein–protein interactions.magnified image

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

“…12 Towards this aim, microfluidic techniques have been described previously for monitoring the denaturation or refolding of proteins by the measurement of extrinsically added fluorophores, 13 or Forster resonant energy transfer (FRET) with fluorescent protein labels. 14,15 The binding affinity between two proteins has also been achieved using two-photon excitation and a fluorescent label. 16 However, the addition of fluorescent labels to proteins can affect their ligand binding, solubility and stability, and it is also difficult to ensure the attachment of only a single label per protein molecule.…”

Basic Technologies for Droplet Microfluidics

“…Various methods have been employed for the detection and characterization of droplet contents. Since most microfluidic devices are fabricated using transparent materials such as PDMS and glass, optical systems, including fluorescence microscopy [53,68,69] and Raman spectroscopy [70], can be directly used to detect the reaction products inside droplets. Up until now, fluorescence measurement has been the most successful and prevalent method to detect the droplet contents.…”

“…Up until now, fluorescence measurement has been the most successful and prevalent method to detect the droplet contents. The advantage of fluorescence detection is that it can simultaneously monitor the fluorescence signal from multiple droplets and has been applied to characterize enzyme kinetics [53] and protein-protein interactions [68] in continuously moving droplets. However, it requires the reaction products to be fluorescent, which limits its applications.…”

Recent advances in microfluidic technologies for biochemistry and molecular biology

“…In recent years, a range of microfluidic tools for performing efficient protein analysis have been reported. For example, protein crystallization (22)(23)(24)(25), protein-protein interactions (26)(27)(28), and two-dimensional gel electrophoresis (29,30) have been realised within planar-chip formats.…”