Dynamic Light Scattering, DLS, to analyse and score Protein Solutions

Sven Falke1, Robin Schubert1, Karsten Dierks2, Markus Perbandt1 and Christian Betzel1

1Institute of Biochemistry and Molecular Biology c/o DESY & The Hamburg Center for Ultrafast Imaging, 22603 Hamburg, Germany

2XtalConcepts, Marlowring 19, 22525 Hamburg, Germany

Dynamic Laser Light Scattering (DLS) is today a well established method to characterize bio-molecular solutions by analysing the dispersity of the suspension and as also recently reviewed by Minton [2016] DLS is the most powerful, highly adaptable and a widely used method to analyse the size distribution of various kinds of particles in solution, till now mostly measuring in cuvettes. Fields of application include size determination and quantification of macromolecules, viscosity determination of blood [Popov and Vitkin, 2016], optimizing solubility and homogeneity of biological samples, analysing dimensions and symmetry of particles [Schubert et al., 2015; Maes et al., 2015; Passow et al., 2015], determining the density of bacterial cultures [Loske et al., 2014], verification of pharmaceutical formulations [Fávero-Retto et al., 2013] support of three-dimensional in vivo imaging, time-resolved analysis of protein assembly or enzyme-catalysed reactions via monitoring changes of the particle size distribution [Georgieva et al., 2004; Yang et al., 2015; Liu and Falke et al., 2016] and monitoring different stages of crystallization reactions [Meyer et al., 2015; Schubert et al., 2017]. DLS is non-invasive and non-destructive and can be adapted to perform measurements in situ in a variety of sample containers, including very thin capillaries to monitor for example counter diffusion crystallization experiments [Oberthür et al., 2012]. In principle, the intensity fluctuations of coherent laser light scattered by particles in solution are recorded over time at a specified angle, correlated with itself after short time intervals and visualized as an intensity auto-correlation function (ACF) [Chu, 1970]. These intensity fluctuations, caused by Brownian motion of particles, are evaluated by algorithms such as CONTIN [Provencher, 1982] and allow to determine the diffusion coefficient of the particles in solution. Considering the viscosity and temperature, the Stokes-Einstein equation is used to calculate the hydrodynamic radius (RH). DLS measurements were successfully used to analyse sample solutions in flow to analyse different stages of protein folding by Gast et al., in 1997 and a particular fiber optic DLS probe was applied by Leung et al. [2006] to characterize latex particles in flow, pointing at a variety of potential industrial applications to count and determine the size of particles for quality control. The application of DLS in a shear flow and in a microfluidic channel was mathematically described by Destremaut et al. [2009], taking the channel dimensions, shear rates, velocity profile of a Poiseuille flow and interferences of different Doppler shifts into account. The resulting theoretical approximation of an ACF with some geometrical restrains underlined that below a critical flow rate the ACF is dominated by Brownian motion of the scattering molecules. In summary, DLS techniques allow to verify the stability and homogeneity of samples in a very time-efficient way and are highly sensitive towards large (unspecific) aggregates of biological macromolecules. This qualifies DLS to be an excellent method for sample quality verification prior or during SAXS experiments. Details and examples will be presented.

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