Microfluidic diffusion analysis of the sizes and interactions of proteins under native solution conditions.

Paolo Arosio, Thomas Müller, Luke Rajah, Emma V. Yates, Francesco A. Aprile, Yingbo Zhang, Samuel I. A. Cohen, Duncan A. White, Therese W. Herling, Erwin J. De Genst, Sara Linse, Michele Vendruscolo, Christopher M. Dobson, Tuomas P. J. Knowles.

ACS Nano 10 333-341, 2015.

In this paper from Arosio et al microfluidic diffusional sizing (MDS) is used in combination with pre-labelled fluorescent biomolecules to size and quantify proteins in complex mixtures. Initially, MDS is compared to Dynamic Light Scatter (DLS) - and a good correlation is observed for monodispersed solutions (Figure 1a). However, the analysis of a polydispersed solution finds that MDS does not exhibit the bias toward larger species seen with conventional DLS. In Figure 1b, DLS measurements overestimate the average size of the mixtures.

The authors note that the measurements using MDS require a significantly lower amount of analyte with respect to DLS, in particular, for species with sizes equal to or smaller than a few nanometers, for which concentrations one order of magnitude higher relative to MDS are needed to generate a detectable scattering signal using DLS.

Comparison of Rh values determined by Microfluidic Diffusional Sizing

Figure1: Comparison of Rh values determined by Microfluidic Diffusional Sizing and DLS in a) monodispersed solutions and b) polydispersed solutions

Finally, MDS is used for the direct detection of specific target species within a complex mixture in a quantitative manner, under native conditions, without the need for separation (as in a Western blot) or for immobilization on a surface (as in an ELISA). To do this the authors use a pre-labelled fluorescent Nanobody against ɑ-synuclein, and observe a size change when incubated with an E-Coli lysate in which ɑ-synuclein is expressed (Figure 2b)

Detection of protein interactions in complex mixtures using MDS.

Figure 2: Detection of protein interactions in complex mixtures using MDS. Change in size of the nanobody in the presence of α-synuclein allows the detection of the binding not only in the homogeneous solution but also in the mixture, where many other proteins are present. Negative controls, represented by lysates where either no protein or a protein that does not interact with the nanobody (in this case the molecular chaperone Hsp70) has been overexpressed, are also shown.

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