LASER DOPPLER ELECTROPHORESIS USING A DIFFUSION BARRIER

Abstract

In one general aspect, an electrophoretic measurement method is disclosed that includes providing a vessel that holds a dispersant, providing a first electrode immersed in the dispersant, and providing a second electrode immersed in the dispersant. A sample is placed at a location within the dispersant between the first and second electrodes with the sample being separated from the electrodes, an alternating electric field is applied across the electrodes, and the sample is illuminated with temporally coherent light. A frequency shift is detected in light from the step of illuminating that has interacted with the sample during the step of applying an alternating electric field, and a property of the sample is derived based on results of the step of detecting.

Claims

1. An electrophoretic instrument, comprising: a vessel, a first electrode, a second electrode, a first diffusion barrier between a sample location and the first electrode, a second diffusion barrier between the sample location and the second electrode, a temporally coherent illumination source positioned to illuminate the sample location, and a frequency-shift detector positioned to receive illumination from the sample location after interaction with the sample.

2. The instrument of claim 1 further including a sample introduction channel to introduce a sample at a sample location in the vessel.

3. The instrument of claim 2 wherein the sample introduction channel includes a needle.

4. The instrument of claim 2 wherein the sample introduction channel includes a port.

5. The instrument of claim 1 further including a sample extraction channel to extract the sample at the sample location in the vessel.

6. The instrument of claim 1 wherein the first diffusion barrier includes a volume of dispersant and the second diffusion barrier includes a volume of dispersant.

7. The instrument of claim 1 wherein the first and second diffusion barriers include a conductive gel.

8. instrument of claim 1 wherein the vessel is a generally upright u-shaped vessel.

9. The instrument of claim 8 wherein the u-shaped vessel further includes a sample introduction port having an opening proximate openings of the u-shaped vessel.

10. The instrument of claim 8 wherein the u-shaped vessel further includes a sample extraction port having an opening proximate openings of the u-shaped vessel.

11. The instrument of claim 8 wherein the u-shaped vessel further includes sample introduction and extraction ports each having an opening proximate openings of the u-shaped vessel.

12. The instrument of claim 1 wherein the vessel is a disposable plastic vessel.

13. The instrument of claim 1 wherein the illumination source is a laser.

14. The instrument of claim 1 further including a zeta potential derivation unit to derive a zeta potential value from an electrophoretic mobility value measured by the detector for the sample.

15. An electrophoretic instrument, comprising: a generally upright, u-shaped vessel, including: a transparent bend, a first upright leg connected to a first side of the bend and extending upward from the bend, a second upright leg connected to a second side of the bend and extending upward from the bend, a first electrode volume connected to an upward end of the first upright leg opposite the bend and extending horizontally away from the first upright leg, and a second electrode volume connected to an upward end of the second upright leg opposite the bend and extending horizontally away from the second upright leg, a first electrode disposed in the first electrode volume and horizontally offset from the first upright leg, a second electrode disposed in the second electrode volume and horizontally offset from the second upright leg, and a cell body for supporting the bend, the first upright leg, the second upright leg, the first electrode volume, the second electrode volume, and the first and second electrodes.

16. The instrument of claim 15 wherein the cell body has a square horizontal cross-section.

17. The instrument of claim 15 wherein the cell body is made of plastic.

18. The instrument of claim 15 further including a temporally coherent illumination source positioned to illuminate a sample location in the bend of the u-shaped vessel, and a frequency-shift detector positioned to receive illumination from the sample location after interaction with the sample.

19. The instrument of claim 15 further including a zeta potential derivation unit to derive a zeta potential value from an electrophoretic mobility value measured by the detector for the sample.

20. An electrophoretic instrument, comprising: a generally upright, u-shaped vessel, including: a transparent bend, a first upright leg connected to a first side of the bend and extending upward from the bend, a second upright leg connected to a second side of the bend and extending upward from the bend, a first electrode at an upward end of the first upright leg opposite the bend, a second electrode at an upward end of the second upright leg opposite the bend, wherein the first upright leg defines a sufficient diffusion barrier volume to prevent dispersion of a sample located in the bend to the first electrode before a frequency shift in light that has interacted with the sample can be detected while an alternating electric field is applied to the sample across the first and second electrodes, and wherein the second upright leg defines a sufficient diffusion barrier volume to prevent dispersion of the sample located in the bend to the second electrode before the frequency shift in light that has interacted with the sample can be detected while the alternating electric field is applied to the sample across the first and second electrodes.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0011] FIG. 1 is a schematic diagram illustrating a prior art electrophoretic measurement;

[0012] FIG. 2A is a schematic diagram illustrating an electrophoretic apparatus according to the invention at the beginning of an electrophoretic measurement;

[0013] FIG. 2B is a schematic diagram illustrating the electrophoretic apparatus shown in FIG. 2A after a first time period has elapsed during the electrophoretic measurement;

[0014] FIG. 2C is a schematic diagram illustrating the electrophoretic apparatus shown in FIG. 2A after a second time period has elapsed during the electrophoretic measurement;

[0015] FIG. 2D is a schematic diagram illustrating the electrophoretic apparatus shown in FIG. 2A after a third time period has elapsed and the electrophoretic measurement is complete;

[0016] FIG. 3 is a schematic diagram of a u-shaped electrophoretic apparatus according to the invention;

[0017] FIG. 4 is a photographic illustration of an implementation of the u-shaped electrophoretic apparatus of FIG. 3;

[0018] FIG. 5A is schematic diagram of a u-shaped electrophoretic apparatus according to the invention that is equipped with introduction and extraction channels, shown before instruction of a sample;

[0019] FIG. 5B is schematic diagram of the electrophoretic apparatus of FIG. 5A shown during introduction of the sample;

[0020] FIG. 5C is schematic diagram of the electrophoretic apparatus of FIG. 5A shown after an electrophoretic mobility measurement for the sample;

[0021] FIG. 5D is schematic diagram of the electrophoretic apparatus of FIG. 5A shown after extraction of the sample for which the mobility measurement was performed;

[0022] FIG. 6 is a schematic diagram of an embodiment of the electrophoretic apparatus of FIG. 5A; and

[0023] FIG. 7 is a plot of concentration against distance for an electrophoretic apparatus according to the invention.

DETAILED DESCRIPTION OF AN ILLUSTRATIVE EMBODIMENT

[0024] Referring to FIG. 2A, an illustrative electrophoretic apparatus according to one aspect of the invention includes a cell 12 with an introduction channel 28, such as a needle. The introduction channel allows a user to introduce a sample, such as a protein sample into a buffer/dispersant 14 at a location that is separated from the electrodes.

[0025] Referring to FIGS. 2A-2D, in operation, the sample cell is filled, initially, only with the buffer in which the sample itself is dispersed in and not the sample itself (see FIG. 2A). The sample is added only to a small region in the vicinity of the detection volume and the measurement started. The measurement proceeds for long enough to record an accurate estimate of the electrophoretic mobility (see FIGS. 2B-2D), but not long enough for the sample to have reached the electrodes. This means that the sample may be retrieved, albeit diluted, afterward without the presence of electrode aggregates.

[0026] Referring to FIG. 3, the electrophoretic apparatus can be based on an upright u-shaped cell 12A in which a sample 16 is injected in an optical detection region at the base of the cell. This provides a relatively long channel length for a given footprint to hold the buffer that acts as a diffusion barrier. The diffusion barrier is intended to isolate the sample (protein, soft sample or otherwise) from the electrode surface whilst maintaining electrical contact with the surface, via the buffer within which the sample is dispersed, for an electrophoretic measurement. In another embodiment, conductive gel plugs, such as agar, which can hinder diffusion further, could be added to the folded cell channel.

[0027] Referring to FIG. 4, the u-shaped electrophoretic apparatus can be implemented as a plastic cell that is compatible with an existing light scattering measurement system, the Zetasizer Nano, which is available from Malvern Instruments Ltd of Malvern, UK, and is described in PCT published application WO2010041082 entitled APPARATUS FOR HIGH-THROUGHPUT SUSPENSION MEASUREMENTS, which is herein incorporated by reference. The Zetasizer Nano can perform different types of measurements, but laser Doppler electrophoretic measurements are the most sensitive for mobility measurements.

[0028] In laser Doppler electrophoretic measurements, the velocity of particles is measured using the technique of laser Doppler anemometry. The frequency shift or phase shift of an incident laser beam caused by the moving particles is measured as the particle mobility, and this mobility can then be converted to a zeta potential of the particles by inputting the dispersant viscosity, and the application of the Smoluchowski or Huckel theories. These theories are approximations useful for most applications. More recent models are available which can give a more exact conversion, but require more knowledge of the chemistry of the dispersion.

[0029] Referring to FIG. 5, a multi-port folded capillary cell 12C can also be used to perform electrophoretic measurements according to the invention. The basic concept is outlined in FIGS. 5A-5D. The cell consist of four ports, two for diluent only (A and B), and two for sample only (C and D). A three-port version would combine the functionality of ports C and D.

[0030] In operation, the whole cell 12C is filled with the buffer within which the sample is dispersed (FIG. 5A). The sample is dropped (pippetted) into the sample cup C and the syringe draws the sample into the measurement chamber (FIG. 5B). The LDE measurement is started immediately. Once the measurement is complete (FIG. 5C), the sample will have diffused someway along the cell arms. Fast field measurements are not affected by electro-osmotic ‘sloshing’ of the sample from electrode chamber to electrode chamber so these chambers are left open at their top and the cell is swept clean by the syringe retrieving the sample, albeit in diluted form (FIG. 5D). Referring to FIG. 6, the multiport cell can also be engineered into a convenient folded form for the Zetasizer Nano.

[0031] Referring to FIG. 7, the diffusion barrier required for a particular measurement can be determined using Fick's first law. Fick's first law describes the concentration, n, at time, t, at a distance x, from a constant concentration source, n(0) and is given by

[00001]$\begin{array}{cc}n\left(x,t\right)=n\left(0\right).Math.\mathrm{erfc}\left(\frac{x}{2.Math.\sqrt{\mathrm{Dt}}}\right)& \left(1\right)\end{array}$

where erfc( ) is the complementary error function. D is the diffusion co-efficient. We focus on lysozyme here with D=120 μm2/s measured using a Zetasizer Nano ZS.

[0032] FIG. 7 shows that many hours need to have elapsed before a lysozyme sample has significantly diffused a distance of 30 mm from the source at x=0. The times taken for protein mobility measurements using micro-electrophoresis are on the order of a few minutes to a few tens of minutes. These are much smaller timescales than shown in FIG. 7. It is likely that convection currents and residual electroosmosis would reduce the times shown in FIG. 7 significantly but it is adequate as a limiting estimate to highlight the theoretical basis for the technique. That is, that LDE measurements can be performed within the time taken for the protein to migrate to the electrode surface if it is required that the sample is retrieved. Or in the time taken for the protein to reach the electrodes plus the time taken for the subsequent protein/electrode aggregates to migrate back from the electrodes to the detection area when it is not required that the protein sample be retrieved intact.

[0033] The present invention has now been described in connection with a number of specific embodiments thereof. However, numerous modifications which are contemplated as falling within the scope of the present invention should now be apparent to those skilled in the art. For example, other cell geometries and injection and/or extraction mechanisms could be devised, and the method could be applied to other types of samples. Therefore, it is intended that the scope of the present invention be limited only by the scope of the claims appended hereto. In addition, the order of presentation of the claims should not be construed to limit the scope of any particular term in the claims.