Patent Description:
Capillary electrophoresis (CE) is an electrokinetic separation method performed in submillimetre diameter capillaries. It is known to integrate capillary electrophoresis on a chip, e.g. using a micro- or nanofluidic channel as capillary, to obtain a miniaturized portable separation solution. Applications of such capillary electrophoresis chips include the separation of biomolecules; for example, for use in mobile point-of-care devices. However, if one wants to reach resolutions in the order of e.g. a single DNA base pair, on-chip capillary electrophoresis poses several challenges. First, a large chip footprint is required because a capillary electrophoresis channel with a length of about <NUM> or more is currently needed to achieve such a resolution. Furthermore, the optimal field for such a resolution corresponds to about <NUM>-<NUM> V/cm; such high voltages are difficult to handle on-chip and particularly in mobile point-of-care devices.

Lower, more manageable voltages can be applied if the capillary is miniaturized. This can be achieved in cyclic capillary electrophoresis, where the capillary forms a closed loop subdivided into sections (e.g. four sections) and the analyte is moved from one section to the next, and thus through the closed loop, by applying an electrical bias over each section in a cyclic fashion. In this manner, an infinite separation channel is realized while allowing the use of lower voltages over the sections. Such a device is for example disclosed in <CIT>.

However, the closed loop implies that the capillary comprises one or more turns. This results in a difference in path length for analytes moving closer to an inner portion of the turn(s) as compared to those moving closer to an outer portion thereof. This is detrimental to the resolution of the method, as it results in continuous broadening and, eventually, overlapping of analyte peaks, so that single base pair resolutions can for example not be achieved.

<CIT> describes a device making use of a first electric field generated by a first electrode pair to induce movement of analytes through a flow path. A portion of the flow path is sandwiched between a first and second electrode of a second electrode pair. Herein, a charge density on the first electrode may be different than on the second electrode.

<CIT> discloses a cyclic capillary electrophoresis device comprising a capillary channel forming a closed loop.

There is thus still a need in the art for cyclic capillary electrophoresis devices which address some or all of the problems outlined above.

It is an object of the present invention to provide good devices for cyclic capillary electrophoresis. It is an object of the present invention to provide good usage associated therewith. This objective is accomplished by devices, methods and uses according to the present invention.

It is an advantage of embodiments of the present invention that peak broadening and the related deterioration in peak separation can be countered. It is a further advantage of embodiments of the present invention that a relatively high resolution can thereby be achieved (e.g. a single base pair resolution for oligomers).

It is an advantage of embodiments of the present invention that the cyclic capillary electrophoresis device can have a relatively small footprint. It is a further advantage of embodiments of the present invention that an efficient and portable point-of-care device can be realized.

It is an advantage of embodiments of the present invention that the cyclic capillary electrophoresis device can be adapted to the operating conditions (e.g. pH) under which it is (expected to be) used. It is a further advantage of embodiments of the present invention that the cyclic electrophoresis device can be adapted on-the-fly to different operating conditions.

It is an advantage of embodiments of the present invention that the capillary electrophoresis device can be fabricated in a relatively straightforward and economical fashion.

In a first aspect, the present invention relates to a cyclic capillary electrophoresis device in accordance with claim <NUM>.

In a second aspect, the present invention relates to a method for forming a cyclic capillary electrophoresis device in accordance with claim <NUM>.

In a third aspect, the present invention relates to the use of a cyclic capillary electrophoresis device in accordance with claim <NUM>.

Similarly, it is to be noticed that the term "coupled" comprises the meaning of the term "connected" but should not be interpreted as being restricted to direct connections only. The terms "coupled" and "connected", along with their derivatives, may be used. Thus, the scope of the expression "a device A coupled to a device B" should not be limited to devices or systems wherein an output of device A is directly connected to an input of device B. It means that there exists a path between an output of A and an input of B which may be a path including other devices or means. "Coupled" may mean that two or more elements are either in direct physical or electrical contact, or that two or more elements are not in direct contact with each other but yet still co-operate or interact with each other. As used herein, and unless provided otherwise, when two entities are said to be "fluidly coupled", it means that a path exists between both entities that allows a fluid (e.g. a liquid) to go from the first entity to the second entity.

As used herein, the "electroosmotic flow" is the motion of electrolyte induced by an applied potential.

As used herein, the term "electroosmotic flow velocity" is the speed of the electrolyte moved by an applied potential.

As used herein, the term "average electroosmotic flow velocity" is the average speed of the electrolyte moved by an applied potential.

We now turn to <FIG> for the purpose of illustration only. The present invention, in general, is described herein with reference to an inner half (<NUM>) facing toward a space (<NUM>) enclosed by the loop (i.e. enclosed by the innermost perimeter of the loop) formed by the channel (<NUM>), the inner half (<NUM>) having an inner wall surface (<NUM>) of first charge density, and to an outer half (<NUM>) facing away from the space (<NUM>) enclosed by the loop formed by the channel (<NUM>), the outer half (<NUM>) having an inner wall surface (<NUM>) of second charge density. The capillary channel (<NUM>) is virtually separated in two halves (<NUM>, <NUM>), the inner half (<NUM>) and the outer half (<NUM>). The channel (<NUM>) has an innermost perimeter (<NUM>) and an outermost perimeter (<NUM>). The innermost perimeter (<NUM>) belongs to the inner half and the outermost perimeter (<NUM>) belongs to the outer half (<NUM>). A virtual perimeter (<NUM>) is defined at mid-distance between the innermost perimeter (<NUM>) and the outermost perimeter (<NUM>) and runs parallel to both perimeters (<NUM>, <NUM>). This virtual perimeter (<NUM>) demarcates the inner half (<NUM>) and the outer half (<NUM>). The inner half (<NUM>) is a part of channel (<NUM>) situated between the virtual perimeter (<NUM>) and the innermost perimeter (<NUM>), while the outer half (<NUM>) is the part of a channel (<NUM>) situated between the virtual perimeter (<NUM>) and the outermost perimeter (<NUM>).

Although the demarcation between the inner half and the outer half can, has it has been illustrated in <FIG>, be placed midway between the innermost perimeter and the outermost perimeter, it is clear that the present invention remains equally valid if this demarcation is placed at any other position along the distance separating the innermost perimeter and the outermost perimeter, except for the innermost and outermost perimeters themselves. Indeed, independently of whether this demarcation is placed closer or farther from midway, the inner half will always be facing toward the space enclosed by the loop and a smaller average electroosmotic flow velocity will always be desired in the inner half in order to compensate for the smaller path length associated therewith.

As used herein, and unless provided otherwise, the term "velocity" refers to "linear velocity".

As used herein, the charge density of an inner wall surface relates only to the density of charges belonging to the material constituting that inner wall or covalently bound thereto and does not relate to charges belonging to the electrolyte or any species transported therein (such as the analyte) that might adsorb on that inner wall surface.

The capillary channel typically contains an electrolyte. The electrolyte is an ionic solution. The electrolyte may be a buffer solution. The electrolyte typically has a sample to be analysed dissolved therein (e.g. in a fraction thereof). The sample to be analysed typically comprises one or more analytes. Examples of analytes are polynucleotide strands, oligonucleotide strands, proteins, peptides, amino acids, and polysaccharides. In embodiments, the cyclic capillary channel may contain a sieving matrix. The sieving matrix is typically a gel. The gel is typically a molecular structure swelled by the electrolyte.

In capillary electrophoresis, the direction of the electroosmotic flow typically depends on the sign of the (surface) charge density of the channel walls. For example, with positively charged walls the electrolyte may flow in one direction, while for negatively charged walls the electrolyte flows in the opposite direction. Moreover, the average electroosmotic flow velocity is proportional to the charge density at the channel wall; i.e. the flow velocity of the electrolyte increases as the walls are more charged and decreases when they are closer to electrical neutrality. It was surprisingly realized within the present invention that this effect can be leveraged, by (e.g. locally) changing the charge density at the channel inner wall surface, to (e.g. locally) tune the average electroosmotic flow velocity. More in particular, the difference in path length in the inner and outer half of a capillary channel can then be compensated for by tuning the ratio between the average electroosmotic flow velocity in the inner half and in the outer half; this is schematically shown in <FIG> depicts the situation for an equal first and second charge density with corresponding equal electroosmotic flow velocities in the inner (VIH) and outer (VOH) halves. This results in a slanted sample peak front after a turn, and thus to intermixing of the peaks and deterioration of the peak separation. However, by increasing the flow velocity in the outer half (<FIG>) or decreasing the flow velocity in the inner half (<FIG>), respectively by increasing the second charge density (depicted as additional negative charges) or decreasing the first charge density (depicted as positive charges neutralizing the negative ones), the migration times of identical analytes in the inner and outer halves can be equalized and the sample peak front can be kept perpendicular to the flow direction.

For determining that a smaller average electroosmotic flow velocity has been created in the inner half (<NUM>) than in the outer half (<NUM>), it suffices to compare the angle made by a pure analyte front with respect to the electrolyte flow after one lap or after one turn in presence of the difference between the first and second charge densities and in absence thereof. If, in presence of the difference between the first and second charge densities, the pure analyte front is now oriented in a direction closer to a perpendicular to the electrolyte flow direction than in absence of that difference, the difference between the first and second charge densities has created a smaller average electroosmotic flow velocity in the inner half than in the other half.

In embodiments, the difference may be adapted for achieving the same migration time to close the loop for an analyte in the inner half than for the same analyte in the outer half.

Preferably, the inner wall surface of the outer half may be negatively charged or can be turned on to become negatively charged. In other embodiments, the inner wall surface of the outer half may be positively charged or can be turned on the become positively charged. Preferably, the inner wall surfaces of both halves may have the same charge sign (i.e. both positive or both negative) or can be turned on to have the same sign, or one may be charged (or can be turned on to be so) while the other is neutral. Most preferably, the inner wall surfaces of both halves are negatively charged or the inner wall surface of the inner half may be neutral and the inner wall surface of the outer half is negatively charged. It is typically not preferred to have oppositely charged inner and outer halves, as this would typically lead to an irregular flow (e.g. because the preferred flow direction in both halves would be opposite).

In embodiments, the difference may be such that the first charge density may be (or may be turned on to be) closer to neutral than the second charge density. Since the path length in the inner half is shorter than the path length in the outer half, in order to bring the migration time in both halves more in line with one another, it is typically advantageous to have a higher charge density for the inner wall surface of the outer half compared to the inner wall surface of the inner half, so as to increase the flow velocity in the outer half compared to the inner half.

There are multiple ways to realize the difference in charge densities. In a first type of embodiments, the cyclic capillary electrophoresis device comprises a first charge-inducing structure capable of being turned on to induce charges on at least part of the inner wall surface of the inner half (and not on the inner wall surface of the outer half), and/or a second charge-inducing structure capable of being turned on to induce charges on at least part of the inner wall surface of the outer half (and not on the inner wall surface of the inner half). In embodiments, the first and/or second charge-inducing structure may comprise (e.g. each comprise) an electrode and a dielectric in-between the electrode and the lumen of the capillary channel wherein the dielectric may be a wall of the capillary channel (in which case the charge-inducing structure is composed of an electrode on a wall of the capillary channel) or a layer provided between the electrode and a wall of the capillary channel (in which case the charge-inducing structure is composed of a dielectric layer on a wall of the capillary channel and of an electrode on that dielectric layer). The charge-inducing structure(s) may operate in a similar fashion as a conventional capacitor (i.e. it may form a capacitive structure), with the electrolyte in the capillary channel acting as a second electrode to realize a typical arrangement of two opposing electrodes separated by the dielectric. This first type of embodiments has the advantage that the charge density and thus the average electroosmotic flow velocity can be adapted after device fabrication (e.g. on-the-fly), thereby allowing to adjust these parameters in function of changes in operating conditions (e.g. a change in the pH of the electrolyte used). However, this flexibility may entail a more complicated device operation, in which the first and second charge densities must still be adjusted prior to or during operation. It may furthermore be advantageous to have both the first and second charge-inducing structures present, thereby allowing to control both the first and the second charge densities and thus better control the average electroosmotic flow velocity in both halves. Nevertheless, it can be sufficient to have only the first or the second charge-inducing structure, thus controlling only the corresponding charge density while keeping the other charge density relatively constant. With a view on equalizing the electroosmotic migration time in both halves, the skilled person can typically determine suitable settings (e.g. a suitable voltage over the electrode) with relative ease through simulations (e.g. computer simulations) or trial-and-error. In embodiments, suitable potential differences between the electrode and the electrolyte may be from <NUM> kV to <NUM> kV.

In a second type of embodiments, at least part of the inner wall surface of the inner half has a first material composition and at least part of the inner wall surface of the outer half has a second material composition, the first material composition differing from the second material composition. When only part of the inner wall surface of the inner half has a first material composition and only part of the inner wall surface of the outer half has a second material composition, the first material composition differing from the second material composition, the part of the inner wall surface of the inner half and the part of the inner wall surface of the outer half preferably belong to a same piece forming the capillary channel. For instance, if the capillary channel is formed of one piece, it is typically sufficient if the part of the inner wall surface of the inner half and the part of the inner wall surface of the outer half both belong to the inner wall of the channel. However, if the capillary channel is formed of two pieces, as is the case when the capillary channel is formed of a substrate comprising a channel having an open top, and of a flat top cover closing the channel, e.g. when the bottom of the channel is etched in a substrate and the top of the channel is provided by bonding the substrate with a top cover, the part of the inner wall surface of the inner half and the part of the inner wall surface of the outer half preferably both belong to the same piece, i.e. both belong to the substrate or to the top cover.

Most preferably, in embodiments, the part of the inner wall surface of the inner half and the part of the inner wall surface of the outer half are present on corresponding locations of respectively the inner half and the outer half.

In embodiments, at least part of the inner wall surface of the inner half or of the outer half may comprise Al<NUM>O<NUM> (e.g. α-Al<NUM>O<NUM>) or TiO<NUM>, and optionally SiO<NUM>, and the other of the inner wall surface of the inner half or the outer half may comprise SiO<NUM>.

In embodiments, at least part of the inner wall surface of the inner half or of the outer half may comprise a self-assembled monolayer bearing charged functional groups or functional groups capable of being charged at a certain pH, while the other of the inner wall surface of the inner half or the outer half may not comprise such a self-assembled monolayer bearing charged functional groups or functional groups capable of being charged at a certain pH. A non-limiting example of suitable self-assembled monolayer bearing a charged functional group capable of being charged at a certain pH is a layer formed from the reaction of (<NUM>-Aminopropyl)triethoxysilane molecules with the inner wall surface. Such a layer is positively charged when in acidic condition. In embodiments, such as for instance in the example of <FIG>, a self-assembled monolayer bearing a positive functional group could form at least part of the inner wall surface of the inner half, while the inner wall surface of the outer half would not be formed of the self-assembled monolayer and would, for instance, consist of SiO<NUM> or TiO<NUM> instead.

For differing first and second material compositions, the corresponding zeta potential at the inner wall surfaces of the first and second halves will typically likewise be different, thereby realizing a difference in charge density between both. Embodiments of the second type have the advantage that, for particular operating conditions, the closed loop nature of the capillary channel can be fully accounted for during device fabrication, without the need to make any changes before or during device operation. A disadvantage of these embodiments may, however, be that they are typically adapted to specific operating conditions and function less well when these are changed (e.g. when an electrolyte of a different pH is used). The difference in zeta potential can in first approximation be linked to a difference in isoelectric point between the first and second material compositions with respect to the operating pH. Nevertheless, it is noted that this is but a first approximation and that a finer approach may in some instances be advantageous in order to accurately estimate the change in charge density that will be obtained for a particular change in material composition. For example, referring to <FIG> of<NPL> in which the zeta potential of α-Al<NUM>O<NUM>, silica (SiO<NUM>), and silica coated with α-Al<NUM>O<NUM> is depicted in function of pH, it can be seen that the zeta potential curves of silica and silica coated with α-Al<NUM>O<NUM> intersect around pH <NUM>. As such, a material composition of SiO<NUM> coated with α-Al<NUM>O<NUM>, as compared to pure SiO<NUM>, would lead to a decrease in charge density (i.e. closer to neutral) at an operating pH above the intersection (e.g. pH <NUM> or more) but an increase (i.e. more negative) below said intersection (e.g. pH <NUM> or less). This conclusion could perhaps not be reached when comparing only the iso-electric point of Al<NUM>O<NUM> and SiO<NUM>. The above notwithstanding, the skilled person can nevertheless determine suitable first and second material compositions in function of a certain device geometry (e.g. angle or curvature of the turns) and operating conditions (e.g. operating pH) with relative ease through simulations (e.g. computer simulations) or trial-and-error.

It will be clear that the first and second type can in embodiments also be combined, i.e. a first and/or second charge-inducing structure may be combined with the inner wall surfaces of both halves having different material compositions. Thus, such embodiments can unite the ease-of-use of the second type with the flexibility of the first type (when needed), but require a more involved fabrication.

The capillary channel forms a closed loop. In embodiments, the outermost perimeter of the loop may be from <NUM> to <NUM>. The height and width (e.g. the diameter) of the (tube forming the) capillary channel are typically uniform along its perimeter. In embodiments, the height and width of the capillary channel may each be from <NUM> to <NUM> to <NUM> to <NUM>.

The charge density of the inner wall surface of the inner half does not need to be uniform. Similarly, the charge density of the inner wall surface of the outer half does not need to be uniform.

In embodiments, the difference between the first and the second charge densities between both halves may be realized by a difference in charge densities only existing or that can only be turned on in a portion of the inner wall surfaces.

The present invention, in general, is described herein with reference to a channel inner half facing toward a space enclosed by the loop formed by the channel, the channel inner half having an inner wall surface of first charge density, and to a channel outer half facing away from the space enclosed by the loop formed by the channel, the channel outer half having an inner wall surface of second charge density, wherein a difference between the first and the second charge densities exists or can be turned on, wherein the difference is adapted for creating a smaller average electroosmotic flow velocity in the channel inner half than in the channel outer half. However, since the channel forms a closed loop due to the presence of one or more turns, the present invention is described with reference to a turn inner half facing toward a space enclosed by the loop formed by the channel, the turn inner half having an inner wall surface of first charge density, and to a turn outer half facing away from the space enclosed by the loop formed by the channel, the turn outer half having an inner wall surface of second charge density, wherein a difference between the first and the second charge densities exists or can be turned on, wherein the difference is adapted for creating a smaller average electroosmotic flow velocity in the turn inner half than in the turn outer half. Hence, any embodiments of the present invention are expressed in terms of turn halves instead of channel halves.

In embodiments, the turn may, but does not need to be, a curved portion of the capillary channel, i.e. a smoothly curving portion of the capillary channel; the turn may however also be an angled portion such as, but not limited to, a right-angled portion.

When the turn is a curved portion of the capillary channel, the portion of the capillary channel belonging to the turn is simply the curved portion forming that turn. In other words, straight channel portions eventually present on each end of a curved portion do not belong to the turn.

When the turn is an angled portion of the capillary channel, there are no non-arbitrary way to define what portion of the capillary channel belong or do not belong to the turn. An angled portion of the capillary channel is an angle between straight portions of the capillary channel, this angle is separated from one or more adjacent turns by two straight portions of the capillary channel. An angled portion will always comprise the angle itself and some length of the two straight portions forming that angle. How much length of these two straight portions forms part of the turn is arbitrary. In embodiments, an angled portion will be considered as consisting of the angle itself and half the length, preferably one third of the length, of each of the two straight portions forming that angle.

In embodiments, the capillary channel may have at least two turns, preferably at least three, such as four turns. A capillary channel with a single turn may, for example, be one with a circular or elliptical shape (i.e. wherein the capillary channel is substantially curved over its whole length) or a shape consisting of a curved portion and a straight portion. A capillary channel with two turns may correspond to a stretched oval shape having two straight portions in the stretched direction (e.g. a 'race track shape'). A capillary channel with three, four, five, six, etc. turns may respectively correspond to a triangular, tetragonal, pentagonal, hexagonal, etc. shape.

In embodiments, the difference between the first and the second charge densities between both halves may be realized by a difference in charge densities only existing or that can only be turned on at one or more turns of the channel inner wall surface. It is at turns that the difference in path length between the inner and the outer halves is created. It is usually more efficient to compensate for the difference in path length created at a turn by modifying the charge densities at that turn, even if it is also possible to compensate for it by modifying the charge densities at other sections of the halves.

When the turn is a curved portion of the capillary channel, the curved portion comprises a first inner wall surface portion at an inner curve (i.e. in the inner half) of the curved portion, the first inner wall surface portion having a first charge density, and a second inner wall surface portion at an outer curve (i.e. in the outer half) of the curved portion, the second inner wall surface portion having a second charge density, and, in embodiments, this is this difference between the first and the second charge densities that either exists or can be turned on.

Expressed differently, in a particular embodiment of the a first aspect, the present invention relates to a cyclic capillary electrophoresis device, comprising a capillary channel having an curved portion, the curved portion comprising a first inner wall surface portion in the inner half of capillary channel, the first inner wall surface portion having a first charge density, and a second inner wall surface portion in the outer half of the capillary channel, the second inner wall surface portion having a second charge density; the device being adapted so that (e.g. in operation) the first charge density differs or can differ from the second charge density (by being turned on), in such a way that an average electroosmotic flow velocity in the inner half of the curved portion is lower than the average electroosmotic flow velocity in the outer half of the curved portion.

When the turn is an angled portion of the capillary channel, the angled portion comprises a first inner wall surface portion in the inner half of the angled portion, the first inner wall surface portion having a first charge density, and a second inner wall surface portion in the outer half of the curved portion, the second inner wall surface portion having a second charge density, and, in embodiments, this is this difference between the first and the second charge densities that either exists or can be turned on.

Expressed differently, in a particular embodiment of the a first aspect, the present invention relates to a cyclic capillary electrophoresis device, comprising a capillary channel having an angled portion, the angled portion comprising a first inner wall surface portion in the inner half of capillary channel, the first inner wall surface portion having a first charge density, and a second inner wall surface portion in the outer half of the capillary channel, the second inner wall surface portion having a second charge density; the device being adapted so that (e.g. in operation) the first charge density differs or can differ from the second charge density (by being turned on), in such a way that an average electroosmotic flow velocity in the inner half of the angled portion is lower than the average electroosmotic flow velocity in the outer half of the angled portion.

Typically, when the average electroosmotic flow velocity in the inner half of a turn is lower than the average electroosmotic flow velocity in the outer half of the turn, this also means that the average electroosmotic flow velocity at any specific distance of the first inner wall surface portion is lower than an average electroosmotic flow velocity farther from the first inner wall surface portion than that specific distance.

The present invention, in general, is described with references to channel halves or to turn halves, and with respect to a charge density difference adapted for creating a smaller average electroosmotic flow velocity in the inner half than in the outer half. However, instead of comparing average electroosmotic flow velocities existing in two halves, one could compare two electroosmotic flow velocities situated at different distances from the innermost perimeter of the channel or from the innermost periphery of a turn of the channel (wherein the innermost periphery of a turn of the channel is comprised in the innermost perimeter of the channel).

For instance, in embodiments, the present invention may relate to a cyclic capillary electrophoresis device, comprising a capillary channel forming a closed loop, the capillary channel comprising:.

Similarly, in embodiments, the present invention may relate to a cyclic capillary electrophoresis device, comprising a capillary channel forming a closed loop and comprising at least one turn, the turn comprising:.

In embodiments, the cyclic capillary electrophoresis device may be a microfluidic device, such as a being, or being integrated into, a lab-on-a-chip (e.g. a mobile point-of-care device). In embodiments, the cyclic capillary electrophoresis device and/or the lab-on-a-chip may have a footprint under <NUM><NUM>. In embodiments, the cyclic capillary electrophoresis device may have a <NUM> base pair resolution, i.e. resolving two oligonucleotide strands differing only by the presence of one additional base pair on one of both strands.

The cyclic capillary electrophoresis device will typically further comprise electrophoretic electrodes for creating a potential difference between separated sections along the capillary channel. In embodiments, the potential difference may be from <NUM> V to 10kV. These electrodes are responsible for the electrophoretic effect on the analytes and electroosmotic effect on the electrolyte. These electrodes are arranged in the device in such a way as to be electrically coupled to the electrolyte when it is present in the capillary channel. In embodiments, these electrodes can be exposed to the lumen of the capillary channel. This permits the electrodes to be in electrical contact with the electrolyte when it is present in the capillary channel. Preferably, they are arranged in a top cover of the device. Typically, at least three such electrodes are used. Preferably, the electrophoretic electrodes are equally spaced along the channel. The electrophoretic electrodes are connectable to a power supply in such a way that a potential difference can be created between non-successive electrophoretic electrodes. In embodiments, the electrophoretic electrodes are connectable to a power supply in such a way that a potential difference can be created between any pair of non-successive electrophoretic electrodes. In embodiments, the electrophoretic electrodes are connectable to a power supply in such a way that a potential difference can be created between any pair of electrophoretic electrodes separated by a single electrophoretic electrode.

Typically, the connection of the electrophoretic electrodes to the power supply can be controlled in such a way that a potential difference can successively be created between different pairs of non-successive electrophoretic electrodes in such a way that the sample travels a complete lap and preferably a plurality of laps around the channel. Hence, in embodiments, the device may comprise a controller electrically coupling the power supply and the electrophoretic electrodes, said controller being adapted to successively create a potential difference between different pairs of non-successive electrophoretic electrodes in such a way that the sample travels a complete lap and preferably a plurality of laps around the channel.

The cyclic capillary electrophoresis device may further comprise reservoirs, formed in the substrate and fluidly coupled to the channel, for storing electrolyte. Each reservoir may be present on an electrical path coupling an electrophoretic electrode and the electrolyte when it is present in the channel. These reservoirs can be used to inject or remove fluid from the capillary channel.

In embodiments, the cyclic capillary electrophoresis device may further comprise a detector for detecting a sample front. The detector may be adapted for evaluating the angle between a sample front and the electrolyte flow direction. In embodiments, one or more windows may be present in the capillary channel for allowing the detector to detect the sample front.

In embodiments, when a first and/or a second charge inducing structure is present, and when a detector is present, the cyclic capillary electrophoresis device may further comprise a controller for automatically adapting the charge density induced by the first and/or a second charge inducing structure to the sample front angle detected by the detector in such a way as to bring the sample front angle to a perpendicular to the electrolyte flow direction.

In embodiments, the cyclic capillary electrophoresis device may comprise:.

Any feature of any embodiment of the first aspect may independently be as correspondingly described for any embodiment of any of the other aspects.

In a second aspect, the present invention relates to a method in accordance with claim <NUM>. The method thus comprises at least one of forming the first charge-inducing structure, forming the second charge-inducing structure, modifying the material composition of the inner wall surface of the inner half and modifying the material composition of the inner wall surface of the outer half; and optionally multiple or all thereof.

The capillary channel formed in step a has its inner wall surface exposed, i.e. it is a groove; it has no top.

In embodiments, the substrate may be a semiconductor (e.g. Si), glass or polymer substrate.

In embodiments, step a may comprise: (a1) etching the capillary channel into the substrate, and (a2) lining the capillary channel with an insulator (e.g. SiO<NUM>).

In embodiments, step b' may be performed before step a and may comprise: (b'<NUM>) etching a cavity in the substrate, (b'<NUM>) forming a dielectric region occupying a first portion of the cavity, and (b'<NUM>) forming a conductive region, on the dielectric region, and occupying a second portion of the cavity. In embodiments, step b'<NUM> of forming the dielectric region occupying the first portion of the cavity may comprise lining the cavity with a dielectric (e.g. SiO<NUM>). In embodiments, step b'<NUM> may comprise filling the cavity with a conductor (e.g. a metal).

In embodiments, step b" may comprise depositing a charge density modifying material onto the inner wall surface of the inner half, selectively with respect to the inner wall surface of the outer half, or onto the inner wall surface of the outer half, selectively with respect to the inner wall surface of the inner half. In embodiments, step b" may comprise depositing a further charge density modifying material onto the inner wall surface of the outer half, selectively with respect to the inner wall surface of the inner half, or onto the inner wall surface of the inner half, selectively with respect to the inner wall surface of the outer half. In embodiments, step b" may comprise altering the material composition of the inner wall surface of the inner and/or outer half (i.e. compared to the corresponding material composition prior to step b"). In embodiments, the altered material composition(s) may have an altered zeta potential. In embodiments, the charge density of the inner wall surface portion of the inner half may be made more neutral (i.e. less charged or closer to neutral than it was originally) and/or the charge density of the inner wall surface portion of the outer half may be made less neutral (i.e. farther from being neutral than it was originally, i.e. more charged, e.g. such as more negative or more positive). In embodiments, the charge density of the inner wall surface of the inner half may be closer to being neutral than the charge density of the inner wall surface of the outer half, wherein both inner wall surfaces have a charge density of same sign or one of both inner wall surfaces has a neutral charge density. In embodiments, the charge density modifying material may have an iso-electric point differing from that of the material composition prior to step b" (but see supra). In embodiments, the charge density modifying material may be Al<NUM>O<NUM> or TiO<NUM>.

Hereafter, a series of six embodiments making use of light and a self-assembled monolayer to enable step b" will be described.

In a first such embodiment, illustrated in <FIG> (left), steps b" and c may together comprise the following steps:.

In a second such embodiment, illustrated in <FIG> (right), steps b" and c may together comprise the following steps:.

In a third such embodiment, illustrated in <FIG>, steps b" and c may together comprise the following steps:.

In a fourth such embodiment, not illustrated, steps b" and c may together comprise the following steps:.

In a fifth such embodiment, illustrated in <FIG> (left), steps b" and c may together comprise the following steps:.

In a sixth such embodiment, illustrated in <FIG> (right), steps b" and c may together comprise the following steps:.

In these six embodiments making use of a self-assembled monolayer and of light to enable step b", one or more of the following may apply:.

In embodiments, step c may comprise anodic bonding of the cover. In embodiments, the cover may be a semiconductor (e.g. Si), glass, quartz, or polymer substrate. In embodiments, the cover may further comprise electrophoretic electrodes, e.g. for inducing an electrophoretic flow.

Any feature of any embodiment of the second aspect may independently be as correspondingly described for any embodiment of any of the other aspects.

In a third aspect, the present invention relates to a use of a cyclic capillary electrophoresis device in accordance with claim <NUM>.

In embodiments, the migration time may be an electroosmotic migration time, i.e. the migration time of the electrolyte. This migration time can, for instance, be for effectuating a lap of the channel or for passing completely a turn (e.g. any turn).

In embodiments, the migration time may be a migration time of any two identical analytes for effectuating a lap of the channel or for passing completely a turn (e.g. any turn).

In embodiments, equalizing a migration time of any two identical analytes for effectuating a lap of the channel, may result in migration times differing by less than <NUM>%, preferably less than <NUM>%, yet more preferably less than <NUM>%.

In embodiments where the capillary channel has at least two turns, an equalizing a migration time of any two identical analytes for passing completely any turn, may result in migration times differing by less than <NUM>%, preferably less than <NUM>%, yet more preferably less than <NUM>%.

In embodiments, any feature of any embodiment of the third aspect may independently be as correspondingly described for any embodiment of any of the other aspects.

It is clear that other embodiments of the invention can be configured according to the knowledge of the person skilled in the art without departing from the true technical teaching of the invention, the invention being limited only by the terms of the appended claims.

A silicon wafer, to be used as a substrate (<NUM>), is first cleaned, e.g. with hot acetone, hot isopropanol and a <NUM> O<NUM> plasma organic cleaning.

We now refer to <FIG>. A first lithographically patterned mask (<NUM>), e.g. with a positive tone photoresist, is provided over the substrate (<NUM>).

We now refer to <FIG>. The first pattern is transferred into the substrate (<NUM>), by etching the substrate (<NUM>) through the openings (<NUM>) defined in the first lithographically patterned mask (<NUM>), thereby defining cavities (<NUM>) with a width of about <NUM> and a depth corresponding to the channel depth.

We now refer to <FIG>. A second lithographically patterned mask (<NUM>), e.g. with a positive tone photoresist, is provided over the substrate (<NUM>), overlapping the first pattern.

We now refer to <FIG>. The second pattern is transferred into the substrate (<NUM>), by etching the substrate (<NUM>) through the openings (<NUM>) defined in the second lithographically patterned mask (<NUM>), thereby defining cavities (<NUM>) with a width of about <NUM> and a depth of about <NUM>.

We now refer to <FIG>. In order to provide enough electrical insulation during voltage application in device operation, the etched surfaces are then passivated by evaporating (e.g. by plasma-enhanced chemical vapour deposition) or thermal growth of a SiO<NUM> dielectric lining (<NUM>).

We now refer to <FIG>. The etched cavities (<NUM>, <NUM>) are filled with a metal (<NUM>), e.g. using autocatalysis, low-melting-point solder or electrodeposition. The metal is for use as an electrode (<NUM>) of the charge-inducing structures (<NUM>, <NUM>).

We now refer to <FIG>. Any excess metal electrode (<NUM>) overfilling the cavities (<NUM>, <NUM>) is removed, e.g. by a chemical-mechanical planarization.

We now refer to <FIG>. A third lithographically patterned mask (<NUM>), e.g. with a positive tone photoresist, is provided over the substrate (<NUM>) for defining a capillary channel (<NUM>) forming a closed loop. The third lithographically patterned mask (<NUM>) is therefore arranged over the substrate (<NUM>) such that the charge-inducing structures (<NUM>, <NUM>) are present at an inner wall of the inner half and an inner wall surface of the outer half of the capillary channel (e.g. such a charge-inducing structures could be present either at each turn of the capillary channel if it comprises two or more turns or along the whole channel length if it comprises only one turn); i.e. a first charge-inducing structure (<NUM>) comprises a first inner wall surface portion (<NUM>) in the inner half and a second charge-inducing structure (<NUM>) comprises a second inner wall surface portion (<NUM>) in the outer half.

We now refer to <FIG>. The third pattern is transferred into the substrate (<NUM>), by etching the substrate (<NUM>) through the opening (<NUM>) defined in the third lithographically patterned mask (<NUM>) and using the metal (<NUM>) as an etch barrier, thereby defining the capillary channel (<NUM>).

We now refer to <FIG>. The substrate (<NUM>) is covered with a SiO<NUM> layer (<NUM>) to provide electrical insulation.

We now refer to <FIG>. The substrate (<NUM>) is then bonded to a cover (<NUM>; e.g. a glass substrate), for example using anodic bonding. The cover (<NUM>) can have been previously furnished (not depicted) with electrophoretic electrodes by depositing <NUM> Au or Pt metal thereon using Cr or Ti as an adhesion layer, followed by patterning the deposited metal to define the electrophoretic electrodes.

Finally (not depicted), the device (<NUM>) can be wire bonded to a printed circuit board for controlling the system.

In the above-described process, two charge-inducing structures are formed: the first in the inner half and the second at the outer half of one of the capillary channel's turn. However, it will be clear that only a single charge-inducing structure (at either the inner or the outer half) could likewise be formed.

We now refer to <FIG>. A lithographically patterned mask (<NUM>), e.g. with a positive tone photoresist, is provided over the substrate (<NUM>) for defining a capillary channel (<NUM>) forming a closed loop.

We now refer to <FIG> The pattern is transferred into the substrate (<NUM>), by etching the substrate (<NUM>) through the opening defined in the lithographically patterned mask (<NUM>), thereby defining the capillary channel (<NUM>).

We now refer to <FIG>. The material composition of select areas of the capillary channel (<NUM>) is then modified, e.g. that of the inner wall surface (<NUM>) in the inner half or that of the inner wall surface (<NUM>) at the outer half. To that end, as a charge density modifying material (<NUM>), for example, Al<NUM>O<NUM> can first be deposited on OH-terminated sites of the substrate (<NUM>) using atomic layer deposition, e.g. through alternate exposures with Al(CH<NUM>)<NUM> (trimethylaluminum or TMA) and H<NUM>O or O<NUM>. The charge density modifying material (<NUM>) is then covered with a photoresist, e.g. deposited by spray coating or spin coating so as to ensure sufficiently good coverage of the entire channel wall. A pattern is subsequently lithographically defined and developed in the photoresist, thereby exposing those portions of the channel wall where the charge density modifying material (<NUM>) is again to be removed. Next, the charge density modifying material (<NUM>) is again removed in said exposed areas, e.g. using tetramethylammonium hydroxide (TMAH or OPD5262).

We now refer to <FIG>. The substrate (<NUM>) is then bonded to a cover (<NUM>; e.g. a glass substrate), e.g. using anodic bonding. The cover (<NUM>) can have been previously furnished (not depicted) with electrophoretic electrodes by depositing <NUM> Au or Pt metal using Cr or Ti as an adhesion layer, followed by patterning the deposited metal.

Example a and example b can also be combined by repeating example a up to and including the step relating to <FIG> and subsequently repeating example b from the step relating to <FIG> onwards.

Claim 1:
A cyclic capillary electrophoresis device (<NUM>) comprising a capillary channel (<NUM>) forming a closed loop, the capillary channel (<NUM>) comprising:
- a turn inner half (<NUM>) facing toward a space (<NUM>) enclosed by the loop, the turn inner half (<NUM>) having an inner wall surface (<NUM>) of first charge density, and
- a turn outer half (<NUM>) facing away from the space (<NUM>) enclosed by the loop, the turn outer half (<NUM>) having an inner wall surface (<NUM>) of second charge density;
characterised in that either:
- a difference between the first and the second charge densities exists, or
- the device comprise:
o a first charge-inducing structure capable of being turned on to induce charges on at least part of the inner wall surface of the inner half, and/or
o a second charge-inducing structure capable of being turned on to induce charges on at least part of the inner wall surface of the outer half,
and the difference between the first and the second charge densities can be turned on by turning on the first charge-inducing structure and/or the second charge-inducing structure,
wherein the difference is adapted for creating a smaller average electroosmotic flow velocity in the turn inner half (<NUM>) than in the turn outer half (<NUM>).