Recent Advances in DLS and DPI Dave Dolak, Malvern Instruments RAMC 2007, San Diego

Classical vs. Backscatter Dynamic Light Scattering (DLS)

Backscatter Optics Dilute Samples Concentrated Samples Backscatter optics facilitate a variable cell position, accommodating both high & low concentration samples. The increased scattering volume ensures high sensitivity, without compromising the working size range or needing to resort to high power lasers which can lead to thermal convection. Scattering Volumes

Advantages of backscatter optics in the Zetasizer Nano instrument Broad concentration range preserves original sample conditions (“Does my sample actually change at lower concentrations?”) Expanded size range (0.6nm - 6um) Enhanced sensitivity yet can handle high concentrations and low concentrations without user intervention/sample prep. Ease of sample preparation  Reduced dust problems: dust/large aggregates scatter light more in the 90-degree and forward direction. (Dust and large aggregates “hosed” 90-degree DLS rendering count rate too variable for measurement.) No so at 173º.  No sample filtration required!

Recent advance: Size and MW from Absolute Size Exclusion Chromatography (ASEC) High resolution from chromatography separation + Ease of use with dynamic light scattering

DPI technology overview DPI stacks two waveguides together--separated by a spacer – which yields an interference pattern at the end of the waveguides. If one waveguide is used as the experimental surface, then changes in the interference pattern can be analyzed to deduce what is happening at the adsorbed layer when various materials are adsorbed to it or as those adsorbed materials change.

DPI Return Thickness Refractive index Density Mass Molar Coverage Volume Fraction Sensing Waveguide Sample Liquid Rest of sensor chip structure including reference waveguide ~100nm DPI offers the ability to quantify changes in the thickness and density (to the sub- Angstrom level) on the chip surface. Sensitive to sub-Angstrom changes in size at picogram concentrations--can detect onset of crystallization at nucleation.

Sensor Surface Å 30 Å Lysozyme pH4 Lysozyme pH7 Sensor Surface Å 45 Å Shape orientation, binding & conformational changes Sensor Surface Dual Polarization Interferometry (DPI) confirms specific binding of Biotin with Streptavidin and resulting conformational change.

Membrane behavior and interactions

The Truth Is Out There DLS and DPI offer new insights into macromolecular behavior.