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Dynamic Light Scattering - an Important Tool for Protein Crystallography and Nanotechnology

Viscotek Europe Ltd.

Dynamic Light Scattering (DLS) is an established technique in the field of protein crystallography. DLS is used to measure hydrodynamic sizes, polydispersities and aggregation effects of protein samples. These are important parameters for the crystallisation of proteins. DLS is also used in the nanotechnology research for the accurate and fast size measurement of nanoparticles made of different materials.

Fig. 1: Schematic setup of a modern DLS-instrument.

Dynamic Light Scattering

Dynamic light scattering, also known as quasi elastic light scattering (Quels) and photon correlation spectroscopy (PCS), measures the laser light that is scattered from dissolved macromolecules or suspended particles. Due to the Brownian motion of the molecules and particles in solution fluctuations of the scattering intensity can be observed.

A modern DLS instrument uses a compact laser diode and high-end fibre optics, so called single mode fibre optic (SMF; Fig.1). The patented SMF optics is the basis for highest sensitivity of DLS instruments. DLS measures the scattered light of a sample in very short time periods and correlates the data. Due to the fact that large molecules or particles move slower than small molecules a defined correlation function results. From the correlation function the diffusion coefficient (D) of the molecules can be calculated by fitting the data. Finally the hydrodynamic radius (Rh) of the particles and molecules can be calculated:

k: Boltzmann-constant

T: temperature

η0: solvent viscosity

The DLS technique is especially impressive because there is no sample information needed. Only the solvent viscosity and the refractive index of the solvent must be known. For nanoparticles the density of the particles might be important. The refractive index increment, an important parameter in static light scattering experiments, is not involved in the calculation of Rh.

Modern DLS instruments are easy to use. The user only has to dissolve or suspend his proteins or nanoparticles (typical concentrations are 0,5-2 mg/ml) and fill it into a microcuvette (cell volume 10-20 µl). Then the hydrodynamic radius of the sample molecules can be determined. Typical time scale for a measurement is less than one minute. A filtration step before measurement is for the majority of samples not needed.

Fig. 2: Correlation function (average from 10 measurements) and calculated hydrodynamic radius for a lysozyme sample. Results: Rh = 1,95 nm, Polydispersity = 19,1%.

Application Example 1: Protein Crystallography

The DLS technique has established itself within the past ten years mainly in the field of protein crystallography. The technique has proved to be a valuable tool for the rapid screening of protein samples for their ability to form crystals. Protein Crystallographers are less interested in the absolute hydrodynamic radius of their samples but more in the polydispersity of the sample and the presence of protein aggregates that might interfere with the crystallisation process.

Fig. 2 shows a correlation function (average of 10 measurements) of a lysozyme sample and the calculated distribution of hydrodynamic radii. Beneath a few small peaks (solvent noise and dust particles) a main peak with 93,8% of scattered light is found that corresponds with a hydrodynamic radius of 1,95 nm. The polydispersity of the sample is 19,1%.

Fig. 2: Correlation function (average from 10 measurements) and calculated hydrodynamic radius for a lysozyme sample. Results: Rh = 1,95 nm, Polydispersity = 19,1%.

The polydispersity is the relative standard deviation of a sample. The polydispersity can be cut into three areas: a sample is monodisperse if the polydispersity is less than 20%, it is medium disperse if the polydispersity is in the range of 20% to 30% and it is polydisperse if the polydispersity is more than 30%.

The lysozyme sample shown in fig. 2 therefore is monodisperse. No protein aggregates are present. Such a DLS result indicates a good chance for crystal growth, but it is not a guarantuee.

An example for a polydisperse sample is shown in fig.3: a small peak is observed with a hydrodynamic radius of 6,21 nm (polydispersity 28,9%). Further broad peaks are visible at 41,4 nm and 212,5 nm and some dust particle in the micrometer range. The chances to get crystals from this protein sample are small. For this sample a filtration step would be necessary to remove the large particles that interfere with the smaller ones.

Fig. 3: DLS result for a inhomogeneous protein sample.

Application Example 1: Nanotechnology

Since several years the DLS technique is used in the field of nanotechnology to measure the size of nanoparticles. Regarding the material properties of the nanoparticles there is almost no limitation; ceramic and metallic/oxidic particles can be measured also as polymer particles and latexes. The size range is from 1 nm to 1000 nm.

Fig. 4: DLS result for a suspension of ceramic nanoparticles in water. Rh = 7,13 nm, polydispersity = 19,8%.

Fig. 4 shows the results for a suspension of ceramic nanoparticles in water. A monodisperse particle species with a hydrodynamic radius of 7,13 nm is found. The following figure shows that DLS can also distinguish different particle sizes in one sample. In this sample two particle species with an Rh of 1,87 nm and 5,31 nm are found. To be able to “separate” two species a radius difference of roughly a factor of 1,5 – 2 is necessary. Another critical point is the concentration ratio of the two or more species. Regarding the fact that a ten times larger particle scatters a million times more light it is obviously that already a small number of large particles will completely cover the scattered light of smaller particles or molecules. This is the reason why sometimes DLS samples has to be filtered to remove interfering dust particles. Once large particles are removed the accurate and reproducible detection of small particles down to 1 nm is possible. The detection limit of the DLS technique is reached when the brownian motion of the sample molecules is the same as the motion of the solvent molecules or when the concentration of sample molecules is too small.

Fig. 5: DLS result for a mixture of 2 nanoparticle species with defined sizes.

Summary

Dynamic light scattering is a powerful technique in the protein crystallography and nanotechnology area. It is possible to measure hydrodynamic radius, polydispersity and the presence of aggregates in protein samples and nanoparticle suspensions. Advantages of modern instruments based on single mode fibre optics are highest sensitivity and short measurement times which is typically less than one minute. Furthermore the use of this instruments and the software has become quite easy so that even non professional users can achieve valuable results. The modern optics often allows to measure samples without previous filtration steps. For this reasons the DLS technique can be used even more effective in the field of protein crystallography and nanotechnology.

 

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