WO2008033021A1

WO2008033021A1 - Moving field capillary electrophoresis with sample plug dispersion compensation

Info

Publication number: WO2008033021A1
Application number: PCT/NL2007/050443
Authority: WO
Inventors: Adrianus Bossche; Florin Tatar; Lujun Zhang
Original assignee: Stichting Voor De Technische Wetenschappen
Priority date: 2006-09-15
Filing date: 2007-09-07
Publication date: 2008-03-20

Abstract

The invention relates to a micro channel system (10) comprising a separation channel (11), a buffer feed channel (12) in fluidic connection with said separation channel (11), and at least two side channels (13, 14) for sample introduction into the separation channel (11), wherein the buffer feed channel (12) comprises at least two electrodes (15, 16) arranged to, during operation, create an EOF in the buffer feed channel (12) so as to produce an additional pressure in the separation channel (11). By producing an additional pressure, a plug profile in the separation channel can be used to create a flat flow profile. By using an additional EOF in the buffer feed channel, there is no need for external pressure source, reducing the size of a microfluidic device incorporating such a micro channel system.

Description

MOVING FIELD CAPILLARY ELECTROPHORESIS WITH SAMPLE PLUG DISPERSION

COMPENSATION

TECHNICAL FIELD

The present invention relates to the use of capillary electrophoresis for compound separation in micro-channels, and more particular to moving field capillary electrophoresis.

STATE OF THE ART

Capillary electrophoresis (CE) is a powerful technique used to separate a variety of compounds. CE analysis is performed by applying appropriate electrical fields to exits of narrow tubes and can result in the rapid separation of many hundreds of different compounds. The separation principle is based on the phenomena that ionized molecules of different mass and charge will travel with different velocities through a capillary under the influence of an electric field. So when a small sample plug is injected at one end of a capillary, the molecules are separated in different zones that can be detected with optical or electrical detectors at the other end. Several attempts have been made to integrate the capillaries in a chip.

Although CE is widely used in laboratory situations for off-line measurement, it has still not found its way to in-line applications in industrial processes, not even after the recent developments of CE microchips. Drawbacks to be overcome for CE micro system integration are the high separation voltages that are required (1500-3000V), fluidic interfacing between process and CE microchip, sample pre-treatment (filtering, enrichment, fluorescence labelling etc.).

An attempt to relieve the barrier for efficient chip integration was the introduction of the Moving Field CE method. In this method, a micro channel contains multiple electrodes and a separation voltage is switched between the electrodes in such a way that it is always applied to that small part of the micro channel (the osmotic region) where the sample plug resides so that the separation voltage can be reduced to a sufficiently low level. However, the flow profile in the osmotic region is largely deformed by a pressure drop in the other sections, which will invoke large sample plug dispersions. In US 6 537 437 Bl micro fluidic devices are disclosed which can be manufactured using surface-micromachining. These devices utilize an electro osmotic force (EOF) or an electromagnetic field to generate a flow of a fluid in a micro channel that is lined, at least in part, with silicon nitride. Additional electrodes can be provided within or about the micro channel for separating particular constituents in the fluid during the flow based on charge state or magnetic moment. The fluid can also be pressurized in the channel.

Publication "Low- voltage driven control in electrophoresis microchips by travelling electric field", Lung-Ming Fu et. al, electrophoresis 2003, 24, 1253-1260 presents the use of a physical model and numerical simulation in the investigation of travelling electric fields on capillary electrophoresis (CE) chips. The principal material transport mechanisms of electro kinetic migration, ionic concentration, fluid flow, and diffusion are all taken into consideration. Traditionally, the high electric field strength required for the separation of biological samples by micro fluidic devices has involved the application of high external voltages. In contrast, this study presents a proposal for samples separation by means of a moving electric field within a low voltage-driven CE chip. The separation channel is partitioned into a series of smaller separation zones by means of electrode pairs.

Publication US 2004/0208751 Al describes a micro channel system comprising a separation channel, a buffer feed channel in fluidic connection with the separation channel, and at least two side channels for sample introduction into the separation channel. The buffer feed channel comprises at least two electrodes arranged to, during operation, create an EOF in the buffer feed channel so as to produce a additional pressure in the separation channel.

A disadvantage in the method descried by US 2004/0208751 Al is that the flow in the sample channel is still pressure driven (although the pressure is generated by an EOF pump). This means that the sample flow has a parabolic flow profile, which will largely distort the profile of the sample plug. For large sample plugs this will be no problem when analysis is done in a connected Mass Spectrometer, however it is not suitable to be used in combination with electrophoretic separation.

SHORT DESCRIPTION It is an object of the present invention to provide a micro channel system which is able to perform CE at low voltages by applying travelling electric fields without the disadvantages of plug distortion due to back pressure.

Therefore, according to an aspect of the claimed invention, there is provided a micro channel system as described above, characterized in that the separation channel comprises a plurality of electrodes arranged to, during operation, create a moving electric field in fluid in the separation channel, the system further comprising a control module arranged to control voltages at the plurality of electrodes of the separation channel, and at the at least two electrodes of the buffer feed channel, wherein a voltage across the at least two electrodes is a function of a voltage applied across two of the plurality of electrodes. Any back pressure distortion generated in the separation channel can be compensated by an extra well-controlled EOF pressure generated in the buffer feed channel. The additional pressure is used to adjust a plug profile of a sample propagating through the separation channel. In this way the plug profile can be flattened. There is no need for an external pressure source. Furthermore, by choosing the right proportion between the voltage applied in the separation channel and the voltage applied in the buffer feed channel any sample plug distortion due to backpressure can be compensated for.

The buffer feed channel may comprise a section between the at least two electrodes that is subdivided in a plurality of smaller channels. By subdividing the section into smaller channels, the wall surface with respect to the total cross-sectional area is enlarged. This will largely increase the EOF driving power, and therefore the applied voltage can be reduced.

In an embodiment, the at least two side channels are in fiuidic connection with each other via a double-T intersection with said separation channel. This configuration is preferred as compared to only one intersection with the separation channel because is optimizes the sample introduction.

The side channels may comprise a section that is subdivided in a plurality of smaller channels. This will create high flow-resistance sections that will minimize flow loss via the side channels during separation.

The invention also relates to a method of electrophoretic separation of particles in a sample, according to claim 6. SHORT DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying schematic drawings in which corresponding reference symbols indicate corresponding parts, and in which:

Figure 1 shows a micro channel system according to an embodiment of the invention;

Figure 2 shows the micro channel system of Figure 1, in which a sample is introduced;

Figure 3 shows the micro channel system of Figure 1 in which the sample is propagating through the separation channel;

Figure 4 schematically shows an example of a channel having a rectangular cross section;

Figure 5 shows a resulting flow profile for the channel of Figure 4;

Figure 6 is an equivalent electrical model of the channel of Figure 4;

Figure 7 schematically shows the channel of Figure 4 including a section across which an additional EOF is applied;

Figure 8 is an equivalent electrical model of the channel of Figure 7;

Figure 9 shows the micro channel system with a control unit according to an embodiment.

DETAILED DESCRIPTION

Figure 1 shows a micro channel system 10 according to an embodiment of the invention. The system 10 consists of a separation channel 11, a buffer feed channel 12 and two side channels 13, 14 for sample introduction. The side channels 13, 14 are fluidly coupled by a double-T intersection. It is noted that the two side channels 13, 14 could also be connected by one cross intersection with the separation channel 11.

The micro channel system 10 comprises a plurality of electrodes 15..25 which are arranged to apply a voltage across sections of the channels 11, 12, 13, 14. The electrodes 15, 16 are arranged to apply a voltage across a section of the buffer feed channel 12. The voltage will create an electro osmotic flow (EOF) in a fluid present in the buffer feed channel 12. The fluid is provided by a fluid buffer which is not indicated in Figure 1. The EOF in this section of the buffer feed channel 12 will be used to compensate a possible EOF profile distortion in the separation channel 11. A profile distortion may for example be the result of a back pressure occurring in the separation channel when applying a moving field separation method as will be explained in more detail below with reference to Figures 6, 7 and 8.

In the embodiment of Figure 1, a section of the buffer feed channel 12 is split up in many smaller parallel channels 30 in order to enlarge the wall surface with respect to the total cross-sectional area. This will largely increase the EOF driving power without the need for high voltages across electrodes 15 and 16.

Each of the side channels 13, 14 comprise a high flow-resistance section 43, 44 respectively. The high flow-resistance section 43, 44 are created by subdividing the side channel 13, 14 into multiple parallel channels. The high flow-resistance sections 43, 44 will minimize a flow loss via the side channels 13, 14 during separation.

In Figure 1 a detection window is indicated with reference number 28. In the detection window 28, a passing sample plug is analyzed using techniques known to the skilled person.

Now, an example of a separation procedure is described with reference to Figure 2 and 3. The separation procedure may comprise the following three steps:

• side channels 13, 14 are filled with buffer (background) fluid

• a sample is introduced in the separation channel 11 via side channels 13, 14

• separation of the sample in separation channel 11 by means of moving field electrophoresis.

Figure 2 shows the micro channel system 10, in which a sample 45 is introduced via the side channels 13, 14 by applying a voltage difference V4 -V₅ on the electrodes 24 and 25. This causes an electro osmotic flow that drags in the sample 45. Voltages at the electrodes 15, 16, 17, 18, 19, 20, 21, 22, 23 may be used to fine-tune the flow such that no sample is introduced in the buffer feed channel or in the separation channel 11 , accept for the double-T intersection.

After the sample 45 has been introduced in the separation channel 11, a voltage difference V2-V3 is applied to create an electric field over the subsection of the separation channel 11 where the sample plug resides. The inner surface of the channel, e.g. made of glass, carries charge. This charge attracts ions in the fluid to the surface until these ions (counter-ions) balance the surface charge. Two layers of counter- ions are distinguished. The Stern Layer which is a thin layer of tightly bound, and thus immobile, counter- ions very close to the surface and a diffuse layer of counter-ions that are only weakly bound. The diffuse layer charges are able to move under influence of electric fields. When a lateral electric field is applied along the channel walls, these ions start to move. Due to viscous forces momentum transfer to the fluid occurs, resulting in the EOF. The Stern Layer plus the diffuse layer are referred to as the 'double layer'.

As the sample plug proceeds through the separation channel 11, the voltages V2 and V3 are switched between subsequent electrodes in such a way that the electric field travels with the sample plug. In Figure 3, the voltage difference is applied across electrodes 17 and 19.

Since the separation voltage is applied to a small section (not more than 1 or a few millimetre), the high electric field required (up to several hundreds of volts per centimetre) can be generated with relative low voltages, for example 20V over lmm yields 200 V/cm.

The other (field- free) parts of the buffer feed channel 12 and the separation channel 11 represent a flow-resistance which introduces a severe parabolic distortion of the desired flat flow profile in the "plug" section.

Figure 4 schematically shows an example of a channel 50 having a rectangular cross section, with dimensions L-d-d where L is the length of the channel 50, and d the thickness of the channel 50.In this example, an EOF flow is generated by means of an electric field in a subsection 51 of the channel 50 having a length α-L (0< α<l). The electric field applied to section 51 causes the double layer to move with a velocity equal to:

with: η = viscosity (water: 10^"3) ζ = zeta potential (0-200 mV) ε = permittivity (ε₀ ^■ ε_r; water ε_r=80) E = electric field (V/cm)

Under free flow conditions (i.e. no pressure difference over section 51) this would yield a EOF flow rate equal to:

Ψ_∞/ = V - Λ (2)

Where A is the cross-sectional area of the channel 50. When the electric field is applied to only the section α of the channel 50 than, due to the flow resistance, a pressure difference Δp develops over the EOF section 51 which causes a parabolic flow rate Ψp in the other (field- free) section(s) and a parabolic back- flow rate (Ψb) in the EOF section 51 of:

Ψ = * and Ψ_b = ^^- (3 4) p R_j1 - (I -OL) - L R_β -a - L ^VJ' ^;

where Ra is the channel flow resistance per unit length for a square cross-section with sides d, the R_H is:

28

R ^Kfl « J — J⁴4 -T ¹iI (5)

Since the mass flow should be constant throughout the channel 50, the pressure over the EOF section 51 can be evaluated from: Ψ_eof - Ψt_>= Ψ_P as:

Ap(a) = Ψ_eof - R_fl - L -a - (l -a) (6)

Figure 5 shows a resulting flow profile with L = 10^"2 m, d = 10^"4 m, α =0.1 and E = 2000V/m. In Figure 5, a curve 54 indicates the velocity of the fluid in the plug in the EOF section 51 across the channel 50, and curve 56 indicates the velocity of the fluid in the field-free regions.

From the previous description an equivalent electrical model can be derived in which the EOF section 51 is represented by a current source ψ_eof with an internal resistance of CcL- R_H, see Figure 6. The field free region can be represented by a resistance having a value of (l-α)-L- R_fl. According to an embodiment of the invention, a plug flow distortion in the separation channel 10 is compensated with an extra pressure difference over the buffer feed channel 12. Using the example of Figure 4, the plug flow distortion can be compensated with an extra pressure difference over the channel 50. This pressure can be generated by an extra osmotic region 52 in the channel 50 having a length of β-L, with (0< β<l-α), see Figure 7. An equivalent electrical scheme of the channel 50 of Figure 7 is shown in Figure 8. The extra osmotic region (i.e. 52) can be modelled by way of a resistance having a value of β-L- Rm with Rm the flow resistance per unit length in the extra osmotic section 52. Assuming identical channel properties in the double layer and the cross-section (i.e. R_H2=R_H), the source Ψ_eof causes a back flow component in section 51 of:

a - L - R -fl

Ψ back, a ⁼ V eof ϊ 7 (V)

-+-

QL - L - R_n (\ -QL ) - L - R_fl + $ - L - R fl2

Likewise, source Ψ_eof2 causes a forward component in section 51 of:

1

V forward, .α ^"Ψso/2 ^' 1 1 (8)

TlT_fl ⁺ $ -L-R_fl2

For a correct plug distortion compensation, both contributions should have the same amplitude. This means that:

^back.α ⁼ ψforward.α (9)

Combining formulas (7), (8) and (9) and using the following substitutions:

it follows that:

with α = relative length of moving field section with respect to separation channel length; β = relative length of buffer feed channel with respect to separation channel; R_fi and Rfl₂ the flow resistance per unit length for the separation channel and buffer feed channel respectively; ζ_α and ζβ the zeta potentials in the separation channel and buffer feed channel respectively; A_α and Aβ the cross-sectional areas of the separation channel and buffer feed channel respectively; V₂- V₃ is the voltage applied across the osmotic region in the separation channel.

If for example α=β=0.1, formula (12) will lead to the conclusion that the voltage over section 52 needs to be 10 times higher than the voltage (and E-field) over section 51. So, when for example E_α= 2000 V/m then E_β= 20000 V/cm.

When section 52 is subdivided in multiple (n) parallel channels (keeping the same overall cross-sectional area). Then the flow resistance of this section changes drastically and can be calculated as follows:

1 12

R '_fli ~ TT^-1I (f°^r rectangular cross-section: w » h) n w- h⁵

When α=β=0.1 and n=10 now only a 1.2 times higher voltage (and E-field) is required over section 52. So, when for example E_α= 2000 V/m then E_β= 2400 V/m. So dividing a section of the buffer feed channel 12 between the electrodes 15 and 16 will significantly reduce the required voltage for producing the additional EOF.

Figure 9 shows a micro channel system 10 comprising the channels described in Figures 1-3. A control unit 60 is present comprising output terminals that are connected to the electrodes 15-25 of the micro channels 11, 12, 13, 14. A detector 62 is arranged to detect a sample in the detection window 28 of the separation channel 11. The detector 62 outputs a signal which is input for the control unit 60. The control unit 60 generates voltages at its output terminals so as to control the Moving Field separation process. The control unit 60 also controls the sample filling by applying suitable voltages to electrodes 24 and 25.

In an embodiment, the control unit 60 is arranged to receive a signal from the detector 62 that indicates when a front of the sample has reached the detection window 28. This is an indication for the EOF properties (e.g. speed) of the separation channel 11 and may be used to fine tune the control of the voltages over the electrodes 17-23 of the separation channel 11.

The voltages required for separation can be derived from the electrical fields and the distance between the respective electrodes 17-23. The distance between the electrodes typically vary between 1-2 mm. The required electrical fields may vary between lOOV/cm and 500V/cm. So, for lmm length, a voltage between 10 and 50 V is needed. For 2 mm distance, a voltage between 20 and 100V is needed.

The invention provides a micro channel system and method that make it possible that a plug flow distortion in a moving field electrophoretic separation channel can be compensated by generating extra pressure in a channel section, such as the buffer feed channel 12. Using parallel sub channels in the buffer feed channel 12, will create an enlarged wall-surface-to-cross-sectional-area ratio. Because of the increase of this ration, no excessive high voltages are required to produce the additional EOF.

While specific embodiments of the invention have been described above, it will be appreciated that the invention may be practiced otherwise than as described.

The descriptions above are intended to be illustrative, not limiting. Thus, it will be apparent to one skilled in the art that modifications may be made to the invention as described without departing from the scope of the claims set out below.

Claims

1. A micro channel system (10) comprising a separation channel (11), a buffer feed channel (12) in fluidic connection with said separation channel (11), and at least two side channels (13, 14) for sample introduction into said separation channel (11), wherein said buffer feed channel (12) comprises at least two electrodes (15, 16) arranged to, during operation, create an EOF in said buffer feed channel (12) so as to produce a additional pressure in said separation channel, characterized in that said separation channel (11) comprises a plurality of electrodes (17, 18, 19, 20, 21, 22, 23) arranged to, during operation, create a moving electric field in fluid in said separation channel, said system (10) further comprising a control module (60) arranged to control voltages at said plurality of electrodes (17, 18, 19, 20, 21, 22, 23) of said separation channel (11), and at said at least two electrodes (15, 16) of said buffer feed channel (12), wherein a voltage across said at least two electrodes (15, 16) is a function of a voltage applied across two of said plurality of electrodes (17, 18, 19, 20, 21, 22, 23).

2. Micro channel system (10) according to claiml, wherein said buffer feed channel (12) comprises a section between said at least two electrodes (13, 14) that is subdivided in a plurality of smaller channels (30).

3. Micro channel system according to any of the preceding claims, wherein said at least two side channels are in fluidic connection with each other via a double-T intersection with said separation channel.

4. Micro channel system according to any of the preceding claims, wherein said at least two side channels (13, 14) each comprise a section that is subdivided in a plurality of smaller channels.

5. Micro channel system according to any of the preceding claims, wherein said control module (60) is arranged to apply a voltage Vi-Vo across said at least two electrodes (15, 16) such that

with α = relative length of moving field section with respect to separation channel length; β = relative length of buffer feed channel with respect to separation channel; Ra and Rfi2 the flow resistance per unit length for the separation channel and buffer feed channel respectively; ζ_α and ζβ the zeta potentials in the separation channel and buffer feed channel respectively; A_α and Aβ the cross-sectional areas of the separation channel and buffer feed channel respectively; V₂- V₃ is the voltage applied across the osmotic region in the separation channel.

6. Method of electrophoretic separation of particles in a sample, comprising:

• filling a separation channel (11) via a buffer feed channel (12) with a background fluid,

• introduction of the sample into said separation channel (11) via at least two side channels (13, 14),

• separation of said sample in said separation channel (11) using a first EOF in said separation channel (11), and

• generation of a second EOF in said buffer feed channel by way of creating an electric field in said buffer feed channel, so as to produce a additional pressure in said separation channel, characterized in that said separation is done by means of moving field electrophoresis using a plurality electrodes (17, 18, 19, 20, 21, 22, 23) comprised in said separation channel (11), said method further comprising:

• controlling of voltages at said plurality of electrodes (17, 18, 19, 20, 21, 22, 23) of said separation channel (11), and at said at least two electrodes (15, 16) of said buffer feed channel (12), wherein a voltage across said at least two electrodes (15, 16) is a function of a voltage applied across two of said plurality of electrodes (17, 18, 19, 20, 21, 22, 23).