Protein Aggregate-Ligand Binding Assays Based on Microfluidic Diffusional Separation

Zhang Y, Buell AK, Müller T, De Genst E, Benesch J, Dobson CM, Knowles TP.

Chembiochem. 2016 Oct 17;17(20):1920-1924

Zhang et al again demonstrate the utility of Microfluidic Diffusional Sizing (MDS) to monitor Nanobody binding to ɑ-synuclein, but this time also use MDS to monitor α-synuclein fibril formation. Due to their size, α-synuclein fibrils diffuse very little and are retained in the centre of the channel, while α-synuclein monomers diffuse more freely. The authors observe that after 40 minutes, fluorescence signal corresponding to the monomeric protein was found to increase in the centre of the channel as a consequence of incorporation into aggregates (Figure 1).

Fluorescence intensity profiles across the channel J) Decrease of free monomer signal outside the bright central region over time during the growth of fibrils. — **Figure 1: Monitoring fibril formation using MDS** i) Fluorescence intensity profiles across the channel. j) Decrease of free monomer signal outside the bright central region over time during the growth of fibrils.

In summary, the method rapidly yields qualitative binding information from only a few microlitres of sample. For quantitative studies, a series of measurements under different conditions enables binding constants and stoichiometries to be established.

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