Recent Advances in DLS and DPI Dave Dolak, Malvern Instruments RAMC 2007, San Diego Classical vs. Backscatter Dynamic Light Scattering (DLS) Backscatter Optics 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 Concentrated Samples Dilute Samples 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 DPI offers the ability to quantify changes in the thickness and density (to the sub- Angstrom level) on the chip surface. Sample Liquid Thickness Refractive index ~100nm Density Mass Sensing Waveguide Molar Coverage Volume Fraction Rest of sensor chip structure including reference waveguide Sensitive to sub-Angstrom changes in size at picogram concentrations--can detect onset of crystallization at nucleation. Shape orientation, binding & conformational changes Lysozyme pH4 pH 4 Lysozyme (pI = 11) Adsorption at 1.0 g dm-3 Lysozyme pH7 pH7 Lysozyme Adsorption at 1.0 g dm-3 (15 uL/min) 6 1.46 7 1.55 5 1.44 6 1.5 1.42 4 5 Refractive Index Response / nm Refractive Index 1.4 and ng/mm2 Response nm 4 and ng/mm2 3 1.45 1.38 2 3 1.36 1.4 2 1 1.34 1 0 1.32 1.35 1890 2390 2890 3390 0 -1 1.3 1210 1310 1410 1510 1610 1710 1810 1910 Time/s -1 1.3 Time/s Sensor Surface 30Å 45Å Sensor Surface Dual Polarization Interferometry (DPI) confirms specific binding of Biotin with Streptavidin and resulting conformational change. Sensor Surface Membrane behavior and interactions The Truth Is Out There DLS and DPI offer new insights into macromolecular behavior.
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