Transcript Recent Advances in DLS and DPI (Dual Polarization

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 subAngstrom level) on the chip surface.
Sample Liquid
Thickness
Refractive index
Density
Mass
~100nm
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
Lysozyme pH7
pH7 Lysozyme Adsorption at 1.0 g dm-3 (15 uL/min)
pH 4 Lysozyme (pI = 11) Adsorption at 1.0 g dm-3
6
1.46
7
5
1.44
6
1.36
1
1.34
4
1.45
3
1.4
2
1
0
1.32
2390
2890
3390
-1
1.3
Time/s
1.35
0
1210
-1
1310
1410
1510
1610
1710
1810
1910
1.3
Time/s
Sensor Surface
Sensor Surface
30Å
45Å
Sensor Surface
Dual Polarization Interferometry (DPI) confirms specific
binding of Biotin with Streptavidin and resulting
conformational change.
Refractive Index
2
Response nm
and ng/mm2
1.38
5
Refractive Index
Response / nm
and ng/mm2
1.4
3
1890
1.5
1.42
4
1.55
Membrane behavior and interactions
The Truth Is Out There
DLS and DPI offer new insights
into macromolecular behavior.