Dynamic Light Scattering (DLS)
Size distributions without fractionation
Dynamic Light Scattering (DLS) measures the translational diffusion coefficients Dt of nanoparticles and colloids in solution by quantifying dynamic fluctuations in scattered light. Sizes and size distributions, in turn, are calculated from the diffusion coefficients in terms of hydrodynamic radius Rh or hydrodynamic diameter dh.
DLS is suitable for ensemble measurements ranging from Rh values of 0.2 nm up to 5,000 nm. Wyatt offers high-throughput, automated DLS as well as conventional, cuvette-based formats.
DLS analysis showing a protein monomer mixed with 50 nm particulates. The size resolution of unfractionated solutions is a factor of 3-5x in radius.
Applications of DLS
Here are just a few of the diverse applications supported by DLS:
- Estimate populations of aggregates large and small, for small molecules, proteins or liposomes
- Check quality of biomolecules prior to running costly analyses such as SPR or SANS
- Analyze thermal stability and chemical denaturation, differentiating pure unfolding from aggregation
- Measure size and concentration of viruses and VLPs
- Quantify self-assembly processes of polypeptides or DNA
- Assess colloidal stability as a precursor to aggregation and precipitation
- Measure viscosity of concentrated biotherapeutics with just a few microliters of solution
DLS determines size distributions without fractionation, providing polydispersity estimates as well as hydrodynamic radii.
Dynamic Light Scattering is also known as Quasi-Elastic Light Scattering (QELS) and Photon Correlation Spectroscopy (PCS).
Automated DLS
Low-volume, automated measurements performed directly in place in standard 96-, 384- or 1536-well microwell plates reveal new horizons for dynamic light scattering. Some of the high-throughput screening tasks accomplished with the DynaPro® Plate Reader include:
- Quantifying type and degree of aggregation in hundreds of buffer conditions, important in formulation and crystallization studies
- Pre-formulation of biotherapeutic candidates to evaluate developability
- Assessment of buffer excipients and pH for colloidal stability using the diffusion interaction parameter kD
- Assessment of multiple formulations for thermal (conformational) stability using the melting temperature Tm
- Screening compound libraries for inhibition or enhancement of protein-protein interactions
- Integration of plate-based testing protocols with other kinds of plate readers
In addition, microwell-plate-based DLS is an attractive option for multi-user labs: with no costly quartz cuvettes to wash, each user can have an inexpensive, personal, disposable plate sufficient for hundreds of samples and no concern for cross-contamination with other experiments.
Melting/aggregation curve data and analyses for four proteins measured simultaneously, in triplicate, on the DynaPro Plate Reader.
Color-coded results of measurements of three proteins, four pH conditions and 6 concentrations, with five replicates per combination, in a 384 well plate. Total measurement time was > 1.5 hours.
Application notes
Selected references
Afonin, K. A.; Kasprzak, W.; Bindewald, E.; Puppala, P. S.; Diehl, A. R.; Hall, K. T.; Kim, T. J.; Zimmermann, M. T.; Jernigan, R. L.; Jaeger, L.; Shapiro, B. A. Computational and experimental characterization of RNA cubic nanoscaffolds. Methods 2014, 67, 256-265.
Akiyama, S. Quality control of protein standards for molecular mass determinations by small-angle X-ray scattering. J. Appl. Crystallogr. 2010, 43, 237-243.
An, W.; Zhang, H.; Sun, L.; Hao, A.; Hao, J.; Xin, F. Reversible vesicles based on one and two head supramolecular cyclodextrin amphiphile induced by methanol. Carbohyd. Res. 2010, 345, 914-921.
Ferré-D'Amare, A. R.; Burley, S. K. Dynamic light scattering in evaluating crystallizability of macromolecules. Method. Enzymol. 1997, 276, 157-166.
Mohr, J.; Chuan, Y. P.; Wu, Y.; Lua, L. H. L.; Middelberg, A. P. J. Virus-like particle formulation optimization by miniaturized high-throughput screening. Methods 2013, 60, 248-256.
Instrumentation for DLS
Cuvette-based DLS Instrument
DynaPro® NanoStar® - With sample volumes as small as 1.25 µL and temperature control spanning -15 °C to +150 °C, the NanoStar goes above and beyond traditional cuvette-based DLS instruments. It offers an optimized static light scattering detector in parallel to the DLS detection system in order to determine true molar mass.
Automated DLS Instrument
DynaPro® Plate Reader - The only commercially available instrument that measures DLS and SLS, for size and molar mass, directly in situ in standard 96, 384 or 1536 microwell plates. Measures second virial coefficient A2 and diffusion interaction parameter kD. The non-perturbing DynaPro Plate Reader offers integration with robot liquid handlers and multi-technique plate-based assay protocols. Temperature-controlled over 4 °C to 85 °C. An integrated camera views each well to observe precipitation or turbidity as well as troubleshooting and diagnostics.
Mobius™ The Mobius is not only the most sensitive zeta potential detector available, it is also a top-of-the-line dynamic light scattering detector. The Mobius provides connectivity to an autosampler for hands off, multi-sample operation.
The Mobius is also the only mobility detector that measures dynamic light scattering (DLS) simultaneously with zeta potential, in the same scattering volume, to monitor potential sample degradation.
On-Line DLS Detector
WyattQELS™ - A dynamic light scattering (DLS) module which integrates into a DAWN, miniDAWN or microDAWN MALS detector to provide simultaneous DLS measurements in the same scattering volume.
Software
ASTRA® - Our comprehensive software solution for MALS and DLS analysis in chromatography, FFF or batch mode. ASTRA is available in a 21CFR(11) compliant version and offers additional options such as particle analysis.
DYNAMICS® - Software for batch DLS measurements in the DynaPro and Mobius instruments, as well as molar mass and A2 in the NanoStar and DynaPro Plate Reader and electrophoretic mobility in the Mobius. Calculates size and size distributions, particle concentration, and derives parameters such as the melting temperature Tm, the aggregation onset temperature Tagg and the diffusion interaction parameter kD.
In the Mobius, DYNAMICS also calculates zeta potential (Henry, Hückel and Smoluchowski) and charge (Debye- Hückel-Henry and effective charge).
DYNAMICS is offered with a 21CFR(11)-compliant Security Pack option, enabling the use of the DynaPro and Mobius instruments in a GMP environment.
Interested in other techniques or combining other techniques with DLS?
SEC-MALS: Standard in protein, biopolymer and synthetic polymer characterizations labs around the world, Wyatt MALS detectors are valued for reliable and robust measurements.
FFF-MALS: Coupling an FFF system to a set of Wyatt MALS and/or DLS detectors creates a powerful system for accurate and robust characterization of molar mass and size distributions for simple or complex samples.
CG-MALS: Apply CG-MALS to characterize self- and hetero-association, binding affinity from pM to mM, absolute molecular stoichiometry (not just mole ratios) and more.
DLS: Measure the translational diffusion coefficients Dt of nanoparticles and colloids in solution by quantifying dynamic fluctuations in scattered light. DLS is suitable for ensemble measurements ranging from Rh values of 0.2 nm up to 5,000 nm.
MP-PALS: Wyatt Technology's breakthrough Massively Parallel Phase Analysis Light Scattering (MP-PALS) extends robust ELS measurements to proteins and other biomolecules in native buffer solutions. MP-PALS does everything conventional zeta potential instruments can do and much more.
SEC-IV: SEC-IV may be the optimal means of characterizing materials not amenable to SEC-MALS analysis. By simply adding a DAWN, transform a basic SEC-IV setup into a powerful SEC-MALS-IV polymer characterization station, to analyze absolute determination of molar mass and size regardless of conformation, branched polymers, copolymers and measure Mark-Houwink coefficients ab initio.