Method and apparatus for focusing charged particles

Abstract

A method of focusing charged particles is disclosed, the method comprising: a) injecting charged particles of at least one substance into a chamber having a major axis; and b) applying an electric field sequence along at least a first portion of the major axis of the chamber, the steps of: b1) applying a first electric field gradient, the first electric field gradient having either a positive or negative sign substantially everywhere within a first region of the major axis and a decreasing magnitude of the electric field along the major axis in the first region of the major axis in a first direction of the major axis; and b2) applying a second electric field gradient, the second electric field gradient having either a positive or negative sign substantially everywhere within the first region of the major axis and a decreasing magnitude of the electric field along the major axis in a second direction of the major axis opposite the first direction.

Description

[Technical Field]

[0001] The present invention relates to the field of manipulating charged particles of matter, and in particular to methods for focusing, i.e., spatially concentrating, such particles. Related methods for carrying out a reaction between at least two substances and for separating at least two substances are also provided. The present invention also provides corresponding apparatus for carrying out such methods. [Background technology]

[0002] The ability to focus a substance so that the volume it occupies is reduced has benefits in a wide range of applications across many industrial sectors. One way a substance can be focused is to form charged particles of the or each substance and then manipulate the position of the charged particles using electric and / or magnetic fields. Optionally, other forces, such as hydrodynamic forces, may be applied. This spatial focusing can be performed on a very small scale, in a so-called microfluidic environment, or on a much larger scale.

[0003] One example of an application in which some degree of focusing occurs is disclosed in International Publication No. WO 2006 / 070176, which describes a method for separating charged objects, particularly by electrophoresis, and an apparatus capable of carrying out such a method. Examples of particles that can be separated in such a process include polymers, such as proteins, DNA molecules, RNA molecules, or other types of biomolecules, such as biological cells. The same principles can be applied to many other types of materials. Here, an electric field gradient is applied to a sample containing a mixture of charged particles disposed in a fluid within a separation channel. The electric field profile is moved along the channel, separating the sample into bands of similar particles, each of which is located at an equilibrium position where the electric force on the particles due to the electric field gradient is balanced by the hydrodynamic force on the particles due to the fluid. The equilibrium position for each band is moved along the channel by controlled movement of the electric field gradient. The shape of the electric field gradient (usually a substantially linear ramp) also leads to a slight narrowing (focusing) of each band as migration progresses. This is because the trailing edge of the band experiences a greater electric force than the leading edge, causing particles at the trailing edge to "catch up" to some extent to particles at the leading edge. However, the focusing achieved is relatively weak, and therefore the different bands at least partially overlap each other during the first part of the separation process for a significant period of time. If the speed of focusing could be increased, this would be advantageous, since a clear separation between the respective bands could be achieved more quickly and over a shorter distance along the separation channel.

[0004] There are many other applications in which enhanced particle focusing is useful, each with correspondingly different advantages. Further examples are provided below. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2006 / 070176 [Patent Document 2] International Publication No. 2012 / 153108 [Non-patent literature]

[0006] [Non-Patent Document 1] Arifuzzaman et al (2006)Large-scale identification of protein-protein interaction of Escherichia coli K-12,Genome Res.16(5):686-91 [Non-patent document 2] Khambhati et al (2019)Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems,Frontiers in Bioengineering and Biotechnology7 [Non-patent document 3] Gregorio et al (2019)A User's Guide to Cell-Free Protein Synthesis,Methods Protoc.2(1):24 [Non-patent document 4] Zhou et al,ChemMedChem,11:738-756,2016 [Non-patent document 5] Louche et al.,2017(Methods in Molecular VBiology,1615:247-255) [Non-patent document 6] Perez et al.,2007(Methods in Molecular Medicine,131:123-139) Summary of the Invention [Means for solving the problem]

[0007] According to a first aspect of the present invention, there is provided a method for focusing charged particles, comprising the steps of: a) injecting charged particles of at least one substance into a chamber having a major axis; b) applying an electric field sequence along at least a first portion of a major axis of the chamber, wherein applying the electric field sequence includes: b1) applying a first electric field gradient, the sign of the electric field being either positive or negative substantially everywhere within a first region of the major axis, the magnitude of the electric field decreasing in the first region along the major axis in the first direction of the major axis; b2) applying a second electric field gradient, the sign of the electric field being either positive or negative substantially everywhere within a first region of the major axis, and the magnitude of the electric field decreasing in the first region along the major axis in a second direction of the major axis opposite to the first direction; A method is provided in which during step (b1), the group of charged particles moves along a major axis in a first direction under the influence of a first electric field gradient, and in step (b2), the group of charged particles moves along a major axis of the chamber in a second, opposite direction under the influence of a second electric field gradient, and during each of steps (b1) and (b2), the leading edge of the group moves slower than the trailing edge of the group due to the smaller electric field magnitude acting on the particles at the leading edge, thereby gradually reducing the width of the group of charged particles.

[0008] By applying this type of electric field sequence to charged particles in a chamber, a significantly greater focusing ratio can be achieved than previously possible, i.e., the width of a group of charged particles can be reduced more quickly. This allows the technique to be used, for example, to achieve a similar proportional reduction in the width of the group as in conventional methods, but over a shorter period of time, or to achieve a greater proportional width reduction given the same length of time. In the first step (b1) of the sequence, the group moves in a first direction, with the trailing edge "catching up" somewhat with the leading edge; then, in the second step (b2) of the sequence, the same group of charged particles moves in the opposite direction, with the trailing edge (formerly the leading edge) "catching up" again. The opposite edges of each group are forced toward each other, which in turn results in a strong focusing (concentration) effect on the entire group of particles. It should be noted that the term "group" is used to describe multiple particles that may be of different types, whereas the term "band" is used to describe multiple particles of the same type (it is likely that such a band will contain particles of one substance, at least in terms of their mobility, but it is not impossible that there may be two or more substances that have particles of the same mobility and therefore remain together in the same band). Thus, a "band" is a more specific type of "group." If the particles injected into the chamber contain particles of different types, the particles will form bands that narrow as a result of the focusing process. The bands may focus at different speeds and may move differently from each other, so that the bands may overlap or be separated from each other.

[0009] As a group of particles exposed to the electric field sequence narrows, it moves back and forth (at least once, preferably more times—in this case, the movement can be described as oscillating) along the major axis of the chamber around some effective mean position. Note that the focusing effect is independent of the presence of any fluid or other sieving matrix (which may or may not be present in the chamber) and does not require the achievement of equilibrium between forces on the particles. Indeed, by switching from a first electric field gradient to a second electric field gradient, any equilibrium condition (if a fluid is present) is typically avoided, or at best, in any one implementation, at any one time, one band of similar particles may feasibly satisfy the equilibrium condition, while other bands of different particle types (due to their different charge / mass ratios) do not. More generally, during the focusing process, no particles are in an equilibrium condition.

[0010] Another consequence of this technique is that the average electric field magnitude experienced by the moving group of particles during step b is non-zero. By switching from the first electric field gradient to the second electric field gradient, particles that moved toward a lower electric field magnitude in step (b1) are then exposed to a higher electric field magnitude (in the opposite direction) in step (b), and vice versa. This results in a non-zero average electric field magnitude (regardless of sign) experienced by the particles throughout step b. Also, the average position around which the particles oscillate has a non-zero electric field magnitude. This leads to a strong focusing effect. Note that the average electric field experienced (or "seen") by the group of particles is not the same as the applied electric field gradient. This is because particles only experience the electric field that exists at the particular position where they are positioned at any one instance, and it takes a finite amount of time to move to another position upon switching the electric field. The electric field magnitude experienced by a group of particles during any particular electric field sequence can be modeled if particle mobility is known.

[0011] The chamber can be of any shape, elongated or non-elongated. Its major axis is the line along which the electric field gradient is applied, and this can have any geometric relationship to the layout of the chamber. However, if the chamber is elongated, the major axis is preferably parallel to the axis of extension of the chamber. The chamber may be a subsection of a larger cavity, which may optionally also contain a separation channel, as described below. The chamber may, for example, be in the form of (all or part of) a cavity formed in the surface of a substrate, and need not be closed on all sides (although this is preferred) provided that it is capable of containing the sample during use. For example, the chamber may have an open top.

[0012] The first portion of the major axis includes all positions on the major axis along which the electric field is applied at some point during the electric field sequence. Thus, at any one time during the sequence, the electric field is applied to all or only a portion of the first portion. The first portion can extend across the entire chamber or only a portion of the chamber. The first region of the major axis is a region of space that is substantially fixed relative to the major axis during at least one cycle of steps (b1) and (b2). Thus, a group of particles disposed in the first region is exposed to a first electric field gradient during step (b1) and then to a second electric field gradient during step (b2). The position and / or lateral extent of the first region may be varied as the method progresses (as described below), but may be varied at a substantially slower pace than the switching between electric field gradients. As described below, in some implementations, there may be one or more additional regions laterally offset from the first region to which other electric field profiles are also applied during the electric field sequence. It will be appreciated that the nature of any electric field profile applied outside the first region during the method is not limited. For example, this may include a zero-crossing point at a location outside the first region, which has little effect on particles in the first region. The possibility of applying an electric field along a direction other than parallel to the major axis during the sequence is also not limited.

[0013] The electric field sequence may or may not include additional steps after (b1) and (b2). In some preferred embodiments, the electric field sequence switches between steps (b1) and (b2) without an intermediate transition step, preferably with substantially no time lapse between steps (b1) and (b2). In other words, the applied electric field switches discretely, preferably instantaneously, from the first electric field gradient to the second electric field gradient (or vice versa if the sequence repeats, as described below). This very sudden change in electric field direction has the advantage that even particles with very high mobilities cannot keep pace with the changing electric field and therefore experience the continuous, strong focusing effect described above. If the switching between steps (b1) and (b2) is too slow, the charged particles may reach and follow the point of minimum electric field magnitude, resulting in a weaker focusing effect.

[0014] However, for many particle mobilities, the instantaneous switching between steps (b1) and (b2) is not necessary. Therefore, in some embodiments, the electric field sequence further includes a transition step (b1') between steps (b1) and (b2), during which the first electric field gradient is changed to a second electric field gradient, and the duration of this transition step (b1') is shorter than the duration of each of steps (b1) or (b2). By keeping the transition step short compared to steps (b1) and (b2), a strong focusing effect can be achieved for a wide range of particle mobilities. In preferred cases, the transition step (B1') is at least 10 times shorter, more preferably at least 50 times shorter, and most preferably at least 100 times shorter than each of steps (b1) or (b2).

[0015] Similarly, the electric field sequence may preferably further comprise a transition step (b2') after step (b2), during which the second electric field gradient is changed to the first electric field gradient, the duration of the transition step (b2') being shorter than the duration of the respective step (b1) or step (b2), preferably at least 10 times shorter, more preferably at least 50 times shorter, most preferably at least 100 times shorter.

[0016] The applied electric field may be changed from a first electric field gradient to a second electric field gradient (or vice versa) in transition steps (b1') and / or (b2'), for example, by translating the electric field profile along a major axis. Each transition step may alternatively or additionally include a period during which no electric field is applied.

[0017] The first and / or second electric field gradients may be stationary during steps (b1) and / or (b2), respectively. However, in a particularly preferred embodiment, during at least a portion of step (b1), the first electric field gradient is translated in a first direction along the major axis of the chamber within the first region, and / or during at least a portion of step (b2), the second electric field gradient is translated in a second direction along the major axis of the chamber within the first region. The entire first or second electric field gradient need not remain within the first region during this translation; a portion of it can move beyond the first region. By translating the electric field gradient in this manner, the particles experience a larger average electric field magnitude (and thus a larger electric force) than if the electric field gradient remained stationary. The reason for this is that the lowest magnitude portion of the gradient (towards which the particles are moving) is itself translated forward in front of the moving particle group, so that the higher magnitude portions of the gradient are always "seen" by the particles more than in the case of a stationary gradient. This further enhances the focusing effect of the electric field sequence. In a preferred case, the translation of the first and / or second electric field gradient occurs for at least half the duration of the respective steps (b1) and / or (b2), preferably throughout the second half of the steps. Most preferably, the translation of the first and / or second electric field gradient occurs throughout the entire duration of the respective steps (b1) and / or (b2).

[0018] The electric field sequence (e.g., step b1 followed by step 2, optionally with transition steps b1' and / or b2') can be performed once. However, in preferred embodiments, the electric field sequence is repeated multiple times, preferably at least three times, more preferably at least 10 times, even more preferably at least 30 times, and most preferably at least 50 times. By repeating the sequence, in effect oscillating between the first and second electric field gradients, the focusing effect is further improved, and thus the amount of focusing is increased. The number of repetitions (oscillations) depends on the desired results, which in turn depends on the application in which the method is deployed. Illustrative examples are provided below. In some cases, more repetitions of the sequence may be desirable, for example, at least 100 times, perhaps up to 200 times. In some applications, there may also be reasons to maintain the narrowed width of the particle group after a desired amount of focusing has been achieved, for example, for a predetermined period of time or indefinitely. Thus, the repetition of the sequence can continue as long as desired, in order to maintain the focused group and avoid diffusion, which would cause the group to widen.

[0019] Repeating the electric field sequence causes the particles to oscillate along their major axes around an average position within the chamber. When multiple different particle types are present, they form bands, all oscillating around the same average position but with different amplitudes depending on their molecular mobility or mass. Each band oscillates and focuses.

[0020] The first and second electric field gradients can each be a different, independent electric field profile, one applied in step (b1) and the other applied in step (b2). However, in some preferred embodiments, as alluded to above, one and the same electric field profile can be utilized to apply both gradients. For example, in step (b), the first and second electric field gradients can each be a compound electric field profile along a first portion of the major axis of the chamber, the compound electric field profile being: a first portion corresponding to a first electric field gradient, the sign of the electric field being either positive or negative at substantially all points and the magnitude of the electric field decreasing in a first direction of the major axis; a second portion corresponding to a second electric field gradient, the sign of the electric field being either positive or negative at substantially all points and the magnitude of the electric field decreasing in a second direction of the major axis opposite the first direction; Applying the electric field sequence includes varying the position of the first portion relative to the first axis by applying a compound electric field profile at different positions along the major axis of the chamber in steps (b1) and (b2), respectively, such that during step (b1), the first portion of the compound electric field profile is applied along a first region of the major axis of the chamber, and during step (b2), the second portion of the compound electric field profile is applied along the first region of the major axis of the chamber.

[0021] This approach simplifies control of the method because only one electric field profile (the composite electric field profile) needs to be stored in memory and can be applied at different locations along the chamber's major axis in each step to achieve the desired effect. It should be noted that such a composite electric field profile typically includes zero-crossing points (i.e., points on the profile with zero electric field magnitude, where the profile has values ​​that vary spatially from positive to negative continuously from one side of the point to the other), but can instead be discontinuous.

[0022] Such a compound electric field profile can be used regardless of whether the sequence includes transition steps (b1') and / or (b2'). If a transition step is included, preferably during transition step (b1'), the first electric field gradient is changed to the second electric field gradient in the first region by moving the compound electric field profile along the major axis. Similarly, during transition step (b2'), the second electric field gradient is preferably changed to the first electric field gradient in the first region by moving the compound electric field profile along the major axis. In both cases, the movement may be translational. In a particularly preferred implementation, the electric field sequence may be repeated multiple times by successively moving the compound electric field profile in alternating directions along the major axis.

[0023] In many implementations, all of the charged particles injected in step (a) have the same charge sign. That is, all of the charged particles injected in step (a) are positively charged, or all of the charged particles injected in step (a) are negatively charged. This may be preferred to simplify handling. In the case of aqueous solutions, the (apparent) charge of the particles can be controlled by controlling the pH of the solution supporting the particles. The apparent charge of particles in solution depends on the isoelectric point of the particles and the pH of the solution. When the pH is set exactly at the isoelectric point, the charge is zero. When the pH of the solution is lower than the isoelectric point, the particles have a net positive charge, and vice versa. Therefore, in some preferred implementations, the pH of the solution supporting mixed types of particles can be selected so that all particles have a positive charge or all have a negative charge. In this way, all particles injected into the chamber move in the same direction as each other in the presence of an electric field and can therefore form part of a group of particles that are focused in the manner described above.

[0024] However, if the injected particles contain particles of different substances, they may simultaneously have a mixture of positive and negative charges. For example, if the pH of the solution is higher than the isoelectric point of a first particle type but lower than that of a second particle type, some of the charged particles will be positive and others will be negative. In such a case, only the first particle type or only the second particle type (but not both) will be focused by the sequential first and second electric field gradients described above. Particles of opposite sign will produce a diffusion effect. If diffusing particles are not of interest, this may not be a problem; therefore, the method can be performed as already described in this scenario, with only a portion of the injected particles forming groups that are focused as the method progresses.

[0025] However, in other preferred implementations, both positively and negatively charged particles can be focused simultaneously in respective groups located in different regions of the chamber along the major axis. This can be achieved as follows: In step b1), simultaneously with applying the first electric field gradient in the first region, a third electric field gradient is applied in a second region of a major axis laterally displaced from the first region, wherein the sign of the electric field is the same as the sign of the first electric field gradient substantially everywhere in the second region, and the magnitude of the electric field increases in the second region along the major axis in the first direction; In step b2), simultaneously with applying the second electric field gradient in the first region, a fourth electric field gradient is applied in the second region, wherein the sign of the electric field is the same as the sign of the second electric field gradient substantially everywhere in the second region, and the magnitude of the electric field increases in the second region in a second direction along the major axis; During step (b1), in the first region, a first group of charged particles having a first charge sign moves in a first direction along the major axis under the influence of a first electric field gradient, while in the second region, a second group of charged particles having an opposite charge sign moves in a second opposite direction along the major axis under the influence of a third electric field gradient; and during step (b2), in the first region, the first group of charged particles moves in a second direction along the major axis under the influence of a second electric field gradient, while in the second region, the second group of charged particles moves in the first direction along the major axis under the influence of a fourth electric field gradient, and during each of steps (b1) and (b2), the leading edge of each group moves slower than the trailing edge of the group due to the smaller electric field magnitude acting on the particles at the leading edge, thereby continuously reducing the width of each group of charged particles.

[0026] Essentially, this preferred implementation of the method involves simultaneously performing the previously described technique in both a first and a second region along a first axis, with the gradient direction being reversed in the second region relative to its direction in the first direction. Particles of a first charge sign (e.g., positive) initially placed in the first region form a first group that converges in the first region, while those initially placed in the second region are diffused (some move into the first region). Meanwhile, particles of the opposite charge sign (e.g., negative) initially placed in the second region form a second group that converges in the second region, while those initially placed in the first region are diffused (some move into the second region). In this way, two groups of particles (one positive, the other negative) are formed, each converging as the method progresses.

[0027] Preferably, the first and third electric field gradients have substantially the same spatial rate of change of the electric field with distance along their major axes relative to each other (but in opposite directions). Similarly, preferably, the second and fourth electric field gradients have substantially the same spatial rate of change of the electric field with distance along their major axes relative to each other (but in opposite directions). In other words, the gradient applied in the second region is substantially a mirror image of the gradient applied in the first region. However, this is not required, and in other cases, it may be appropriate to apply gradients of different slopes to each region, for example, when particles of one charge sign are known to have significantly different mobilities from particles of the opposite charge sign.

[0028] The first and second regions are each substantially fixed relative to the major axis, but as described above with respect to the first region, either or both can move and / or change size slowly relative to the steps of the electric field sequence. The first and second regions are separate regions of space and do not overlap. The first and second regions can be adjacent to each other, but preferably have a non-zero space between them, forming a third region that is preferably smaller than either the first or second region. The size of the third region can also be changed as the method progresses (at a slower pace than the steps of the sequence). Between the two regions where particle focusing occurs, the third region acts as a buffer zone, advantageously reducing the likelihood that particles of the first group will move out of the first region and reach the second region (where they have the opposite charge sign to the second group, causing them to be scattered and thus lost from the first group). The third region can be a region to which no electric field is applied during the electric field sequence. However, in a more preferred embodiment, a non-zero electric field may be applied in the third region, preferably configured to cause the particles to move again towards their respective groups. Because both positive and negative particles can move into the third region, mixing (and optionally reaction) of such particles can occur here.

[0029] Thereby, preferably, in step b1), simultaneously with applying the first and third electric field gradients, a non-zero electric field of the same sign as the signs of the first and third electric field gradients is applied in the third region, the non-zero electric field preferably having a spatial rate of change of the electric field as a function of distance along the major axis that is smaller than that of the first and third electric field gradients, most preferably substantially constant across the third region; In step b2), simultaneously with applying the second and fourth electric field gradients, a non-zero electric field of the same sign as the second and fourth electric field gradients is applied in the third region, the non-zero electric field preferably having a spatial rate of change of the electric field as a function of distance along the major axis that is smaller than that of the second and fourth electric field gradients, and most preferably is substantially constant across the third region.

[0030] By arranging the non-zero electric field in the third region so that it has the same sign as the gradients on either side of it at each step of the sequence, negative particles that reach the third region are moved in one direction along the major axis (towards the region where the group is focused), and positive particles are moved in the opposite direction. This reduces particle loss from each group and thus enhances the focusing effect. In a preferred embodiment, the non-zero electric field in the third region has a spatial rate of change of the electric field as a function of distance along the major axis that is smaller (i.e., shallower slope) than that of the gradients on either side of it, and most preferably flat (substantially constant). This means that any focusing or diffusing effect of the electric field in the third region is weaker than in the first or second regions, and preferably non-existent, making the region substantially charge sign agnostic; i.e., neither positive nor negative particles experience any significant diffusing effect in the third region.

[0031] It should be noted that although the charge sign of the particles is described above as being dependent on the pH of the solution in which the particles are supported, the method does not necessarily have to be carried out in solution. The method can be implemented, for example, as a gas phase system, in which case the charged particles can be generated by ionizing one or more substances. This usually results in all positively charged particles (however, this depends on the process by which the ionization is carried out).

[0032] Preferably, the first and second electric field gradients have substantially the same spatial rate of change of the electric field as a function of distance along their major axes relative to each other. That is, the slope of the first electric field gradient applied in step (b1) in the first region is substantially the same as that of the second electric field gradient subsequently applied in step (b2) in the same region. This produces a symmetric focusing effect, causing the group of particles in the first region to oscillate about a mean position approximately in the center of the first region. In other cases, the slopes of the two gradients can be different from each other if a less symmetrical result is desired. When third and fourth successive electric field gradients are applied to the second region as described above, it is also preferred that the third and fourth electric field gradients have substantially the same spatial rate of change of the electric field as a function of distance along their major axes relative to each other for the same reasons.

[0033] It may also be desirable for the first and second electric field gradients to have substantially the same maximum and minimum electric field magnitudes, so that the particles are exposed to the same electric forces (but in opposite directions) at each step, which also contributes to symmetrical oscillations of the particle groups. The same considerations preferably apply to the third and fourth electric field gradients.

[0034] Similarly, it is preferred that the first and second electric field gradients have substantially the same profile shape as each other, so that the difference between the electric field experienced by the particles at the leading edge of the group and at the trailing edge of the group is substantially the same in steps (b1) and (b2), which improves the focusing effect. The same requirement preferably applies to the third and fourth electric field gradients.

[0035] The electric field gradient can have a variety of shapes, each with the magnitude of the electric field being greater at one end of the gradient than at the other. Preferably, however, the first and / or second electric field gradients are monotonic (i.e., continuously increasing or continuously decreasing in a spatial, but not temporal, sense) at least along a first region of the major axis. In preferred examples, the first and / or second electric field gradients are substantially linear or curvilinear, such as parabolic or exponential. It will be appreciated that at very small scales, the electric field may be a stepped approximation to such a shape as a result of the individual properties of some electric field generating means (e.g., electrode arrays). All of these preferred features apply equally to the third and / or fourth electric field gradients in the second region.

[0036] It is preferred that steps (b1) and (b2) have substantially the same duration as each other. This has the result that the particles are subjected to opposing forces for substantially equal durations, which helps to achieve a more symmetrical focusing effect when the first and second electric field gradients have substantially the same slope. Although this is not essential, the durations of the two steps should be at least of the same order of magnitude as each other.

[0037] It will be appreciated that the electric field sequence can potentially include one or more additional steps without compromising the overall focusing process described above. Preferably, however, steps (b1) and (b2) together form the major part of the sequence, with any additional steps representing a minor portion of the process time. In the preferred case where the electric field sequence is repeated multiple times, steps (b1) and (b2) together occupy at least 80%, preferably at least 90%, and more preferably at least 99% of the duration of each cycle. (By "cycle" it is meant that all steps in the sequence are performed once in order.)

[0038] In some particularly preferred implementations, the electric field sequence may consist of steps (b1) and (b2) plus optional transition steps (b1') and / or (b2'), and the electric field sequence is preferably repeated multiple times. In other words, the electric field sequence preferably includes only step (b1) followed by step (b2), or preferably includes only steps (b1), (b1'), (b2), and (b2'), in that order, with no additional steps in the sequence.

[0039] The cycle time depends on the desired results and the mobility of the particles involved. In a preferred embodiment, the electric field sequence has a total duration (i.e., cycle time) of 0.1 to 100 seconds, preferably 0.1 to 10 seconds, more preferably 0.1 to 5 seconds, and most preferably 1 to 3 seconds. For different applications of the method, different cycle times are suitable, examples of which are provided below. The cycle time can be set by the user via control of the electric field applied to the chamber.

[0040] The amount of particle movement experienced during the focusing method depends, among other things, on the particle mobility, the magnitude of the electric field, and the duration for which the various electric fields are applied. This can be predicted by computer modeling of the process, and various parameters can be adjusted, if necessary, to achieve the desired amount of movement, i.e., the spatial extent of particle oscillation. The desired oscillation width will depend on the application. In some preferred embodiments, the electric field sequence in step (b) is repeated multiple times, with the first and second electric field gradients and the duration of each cycle configured to oscillate the charged particles along the major axis of the chamber, and the oscillation width ranges from 0.1 to 10 mm, preferably 0.5 to 5 mm, and more preferably 1 to 3 mm. The oscillation width can be controlled to remain substantially constant over the duration of the method or to narrow as the method progresses, for example, by (preferably gradually) shortening the cycle time. Narrowing the oscillation width during the method reduces the time required to achieve a given level of group focusing.

[0041] Vibration of the first group of particles occurs in a first region of the major axis of the chamber (i.e., where the first and second electric field gradients are sequentially applied). In some preferred embodiments, the first region of the major axis of the chamber has a length (along the major axis) of 0.1 to 5 cm, preferably 0.5 to 2.5 cm, and more preferably 0.5 to 1.5 cm. The second region (if present) may also have a length in the same range.

[0042] Although several examples of oscillation amplitudes and area dimensions are provided above, in fact, the usefulness of the disclosed method is not limited to any particular scale. The above examples are primarily suited to small scales, e.g., microfluidic implementations, so that the focusing method may be used in scenarios where it is part of a diagnostic test or analysis involving, for example, living cells. However, in other applications, the technique can be employed at significantly larger scales, for example, when reacting large amounts of materials to manufacture products in industrial settings. The physical configuration of the device and the manner in which the electric field is generated may vary depending on the scale (as described below), but the principles of the method remain the same.

[0043] Similarly, the nature of the charged particles injected in step (a) depends on the application in question. In some cases, the particles can all be of the same type, for example, if the sole purpose is to increase the concentration of a substance (i.e., to reduce the volume occupied by the amount of substance). However, in many applications, a mixture of particles is present, and the purpose is to react and / or separate different types of particles. Therefore, preferably, in step (a), the charged particles are of multiple substances, including charged particles of different mobilities, and during step (b), the group of charged particles is additionally formed into multiple bands under the influence of an electric field sequence, each band including charged particles with the same mobility, and the width of each band is successively reduced. Thus, both the individual bands and the entire group of particles (i.e., the set of bands) are focused as the method progresses, by the mechanisms already described.

[0044] As noted above, the first region (and second region, if provided) are generally fixed relative to the major axis during at least one cycle of the sequence, although it is possible to vary the size and / or position of the region over a longer time frame. Thus, in some preferred embodiments, in step (b), the electric field sequence is repeated a plurality of times, and the size of the first and / or second region is reduced as the method progresses by reducing the width of the major axis along which the respective electric field gradient is applied at least once between successive cycles, preferably every N cycles, where N is an integer greater than or equal to 1. Most preferably, N is greater than 1. By reducing the size of the or each region as the method progresses, the oscillation width can be reduced, further enhancing the focusing effect.

[0045] Furthermore, at the beginning of the process, the injected particles (or "plug") may be spread over a relatively large volume within the chamber, which may be several centimeters long. In such cases, if the oscillations are performed where the first and second electric field gradients extend over only a few millimeters, this may "crop" the injected plug, leaving most of the injected sample substantially outside the oscillations.

[0046] Therefore, the electric field gradient may initially need to be applied over a relatively wide area to capture a sufficient number of particles. As the method progresses, the particles focus together, which can correspondingly reduce both widths. Thus, in some implementations, step (b) may include a particle collection stage and a subsequent particle focusing stage, where the size of the first and / or second areas is larger in the particle collection stage than in the particle focusing stage. The particle collection stage may include one cycle or multiple cycles in sequence. However, typically, the number of cycles in the particle collection stage is smaller than the number in the subsequent particle focusing stage.

[0047] For the same reason, it may be desirable to selectively vary the slope of the electric field gradient as the method progresses to coincide with any changes in region size. In a preferred case, the spatial rate of change of the electric field magnitude as a function of distance along the major axis of each electric field gradient is increased as the method progresses, preferably lower in the particle collection phase than in subsequent particle focusing phases. For example, in the initial cycles of the sequence, the gradients of the first and second electric fields may be relatively shallow (and selectively applied over a wider region) to trap particles and apply a weak focusing effect to begin collecting them. The gradients may then be increased (and selectively narrowed) to strengthen the focusing effect and achieve the desired focusing of the trapped particles. In one example, there may be one oscillation period in which a wider electric field is applied, occupying approximately 5 cm and resulting in an oscillation of approximately 1.5 cm. This single back-and-forth step can shrink the initial 1 cm plug to approximately 5 mm wide. Subsequent repetitions of the electric field sequence may be performed over a significantly narrower first region to achieve the desired strong focusing effect.

[0048] Other methods of trapping particles before focusing begins are possible. For example, in another embodiment, the method further comprises, after step (a) and before step (b): (a') collecting the charged particles to position a majority, preferably substantially all, of the group of charged particles within a first portion of the major axis of the chamber.

[0049] Preferably, step (a') comprises: a'1) applying an electrostatic field gradient along a second portion of the major axis that is larger than and includes the first portion, the electrostatic field gradient including a zero-crossing point origin located in the first portion, and the charged particles moving toward the zero-crossing point under the influence of the electrostatic field gradient.

[0050] This achieves a weak focusing effect that moves the particles into the first portion of the chamber so that they are exposed to the electric field sequence applied in step (b). For example, this collection step can include the application of a wide, shallow electric field gradient that is static and crosses zero near the center of the future oscillation. If desired, as the repetitions of step (b) progress, it is possible to include particle collection step (a') and then narrow the region and / or increase the steepness of the electric field gradient.

[0051] A first aspect of the present invention is an apparatus for focusing charged particles, comprising: a chamber into which, in use, charged particles of at least one substance are injected, the chamber having a major axis; an electric field generator configured to apply an electric field along at least a first portion of a major axis of the chamber; a controller configured to control the electric field generator and programmed to control the electric field generator to apply an electric field sequence along at least a first portion of the major axis of the chamber, the electric field sequence comprising: b1) applying a first electric field gradient, the sign of the electric field being either positive or negative substantially everywhere within a first region of the major axis, the magnitude of the electric field decreasing in the first region along the major axis in the first direction of the major axis; b2) applying a second electric field gradient, the sign of the electric field being either positive or negative substantially everywhere within a first region of the major axis, and the magnitude of the electric field decreasing in the first region along the major axis in a second direction of the major axis opposite to the first direction; The present invention further provides an apparatus wherein, in use, during step (b1), the group of charged particles moves along a major axis in a first direction under the influence of a first electric field gradient, and in step (b2), the group of charged particles moves along a major axis of the chamber in a second, opposite direction under the influence of a second electric field gradient, and during each of steps (b1) and (b2), the leading edge of the group moves slower than the trailing edge of the group due to the smaller electric field magnitude acting on the particles at the leading edge, thereby gradually reducing the width of the group of charged particles.

[0052] The apparatus provides all the benefits of the methods already described. Suitable examples of the physical configuration of the apparatus include the electrophoresis apparatus disclosed in WO 2006 / 070176 or WO 2012 / 153108, although the control device is of course programmed differently in the manner specified above. Preferably, the control device is further programmed to control the electric field generator to perform any of the preferred implementations of the methods described above.

[0053] The electric field generator can take any form that allows for the application of electric field gradients of the types described along the major axis of the chamber, and for changing from one electric field gradient to another. In a preferred embodiment, the electric field generator includes an array of electrodes arranged along at least a first portion of the major axis of the chamber. In use, appropriate voltages are applied to each electrode to produce the desired electric field gradient. The electrode array is particularly well suited for generating electric field gradients in small-scale (e.g., microfluidic) implementations of the device. The electrode array can have the same general form as disclosed, for example, in WO 2006 / 070176 or WO 2012 / 153108.

[0054] Preferably, the chamber is provided with at least one input port for injecting a sample into the chamber, the sample comprising at least charged particles of at least one substance. Desirably, the chamber is provided with at least one exit port for extraction of groups or bands of charged particles from the chamber. Additional electrodes may be associated with the input and / or exit ports to allow application of additional electric fields to inject particles into or extract particles from the chamber.

[0055] In a preferred embodiment, the apparatus further comprises a detector adapted to detect groups or bands of charged particles in the chamber, the detector preferably being adapted to image the groups or bands.

[0056] As noted above, the disclosed method for focusing charged particles has applications in many applications and industries. A second aspect of the invention relates to one such term.

[0057] Accelerating the rate of certain reactions (the term "reaction" is used herein to encompass both chemical reactions and biological interactions, such as binding events) is critical to many areas of science and industry. For example, catalysts are used to convert heavy oil into gasoline or jet fuel. Reducing incubation times in immunoassays substantially impacts the speed and throughput of important diagnostic tests for infectious diseases, dementia, cancer, and cardiovascular disease. These are just a few examples.

[0058] Traditionally, there are four ways to increase the reaction rate: 1) Use of Catalysts Over 80% of the chemical industry uses catalysts to increase the rate of reactions. 2) Increase in temperature Increasing the temperature increases the average velocity of the particles and therefore the reaction rate. 3) Increasing the concentration of reactants Increasing the concentration increases the chances of collisions between particles and therefore the reaction rate. 4) Increased Surface Area of ​​Reactants Increasing the surface area of ​​a particle increases the probability of reaction for any given molecule.

[0059] As an alternative to (2), electrokinetic mixing has also been proposed. This involves applying an alternating current to the fluid to cause localized heating and thus buoyancy-driven flow of the fluid, resulting in mixing. Techniques for further accelerating reactions are desirable.

[0060] Accordingly, a second aspect of the present invention is a method for causing a reaction between at least two substances, comprising the steps of: (i) providing charged particles of at least two materials; (ii) focusing charged particles in a chamber using the method of the first aspect of the invention, wherein the charged particles of each substance form respective bands, the widths of the bands being successively reduced, and the respective bands overlapping each other during at least a portion of the electric field sequence, thereby promoting reaction between the substances.

[0061] Thus, the reaction method uses the above-described charged particle focusing method to accelerate a reaction between at least two substances supported in a liquid or present as a gas. As noted above, the term "reaction" (and related words, such as "react") herein refers to chemical reactions and biological interactions, such as binding events. This technique brings particles of different substances together in the same spatial location over a predetermined period of time, increasing the concentration (i.e., amount of substance per unit volume) of each substance. As a result of both effects, the reaction proceeds significantly faster than previous methods. Depending on the properties of the various particles (e.g., their respective mobilities) and the electric field parameters, the respective bands of particles may cross each other as they vibrate in the chamber, or may simply occupy the same (progressively smaller) volume. The proximity of different particles increases the probability of collisions, thus promoting the reaction. Additionally, the reaction may also be enhanced by the increased average velocity (and therefore momentum) of the particles due to the vibration, resulting in increased collisions when bands overlap each other in the chamber. In tests, the disclosed method was successful in very substantially reducing the time taken for reaction processes, including SARS-CoV-2 antigen tests, Lassa fever tests, and tests for influenza viruses.

[0062] An additional benefit of the disclosed methods observed in some cases is a significant increase in reaction efficiency. That is, the amount of analyte required for a successful reaction is significantly reduced. This, in turn, uses fewer resources and reduces costs. For example, in the case of immunoassays, a probing antibody (or antigen) is used. In conventional assays, the target analyte (antigen) is typically expected to be in low concentration (or rather, the sample of interest is one with a small amount of target molecule. For example, in COVID-19 antigen testing, early detection is important, where antigens are present in trace amounts). To ensure incubation (i.e., reaction) occurs, a very high concentration of probing antibody (probing antigen in the reverse reaction) is typically used to ensure that each target antigen finds a matching antibody to react with. Low antibody concentrations result in a small chance of matching and extremely slow incubation rates. Using conventional techniques, it is also typically necessary to incubate the sample before chemical analysis is performed to increase the amount of target antibody / antigen complex present to reach a detectable amount. Sufficient incubation may take several hours or even days.

[0063] However, because the disclosed method essentially concentrates and collides the probing and target molecules, the starting amount of the probing analyte can be significantly reduced. Even at very low initial concentrations when injected, both the probing and target molecules are concentrated by the electric field sequence, and repeated collisions can result in higher reaction efficiency than conventional incubation, even when the probing molecule is at a very high concentration in conventional incubation. For example, the inventors discovered that for a SARS-CoV-2 antigen test in which the target molecule is the nucleocapsid protein, a concentration on the order of 500 μg / μL was required to achieve a 1-hour incubation using conventional techniques. Incubation was volatile, with a significant failure rate. In contrast, using the disclosed method, without conventional incubation, the probe concentration was reduced to 50 μg / μL, yet a significantly higher signal was achieved from the reacted complex (Ab+Ag).

[0064] It will be appreciated that although the benefits of the reaction method are described with reference to an immunoassay scenario, the same principles apply to all types of chemical (or biochemical) reactions.

[0065] The method may include any of the preferred features of the first aspect of the invention described above. In connection with carrying out a reaction, it is preferred that particles of all substances required to react are arranged to have the same charge sign, so that the resulting bands are focused in the same region of the chamber. It is possible to react particles of opposite sign (because particles of opposite sign will be mixed in some parts of the chamber), but this will be less efficient.

[0066] Preferably, to further promote the reaction, the electric field sequence is repeated multiple times, resulting in many oscillations of the bands (and thus more collisions). For example, in some advantageous implementations, in step (ii), the electric field sequence is repeated multiple times, preferably at least 30 times, more preferably at least 50 times, and even more preferably at least 100 times. Alternatively or additionally, the electric field sequence can be repeated (i.e., oscillations are continued) until the reaction has progressed by a desired amount. For example, in step (ii), the electric field sequence can be repeated until a predetermined amount of one of the at least two substances is reached, and the predetermined amount is preferably 30%, 50%, 70%, 80%, 90%, 99%, or 100%. The percentages here refer to the proportion of the analyte that has reacted. 100% corresponds to the stage where all molecules of one of the reactants have reacted. The percentages refer to the primary reactant (substance), i.e., one that ideally reacts 100%. To increase the amount of primary reactant that actually reacts, an extra secondary reactant may be added. For example, in an antigen test, the primary reactant is an antigen and the secondary reactant is an antibody. One or more runs of the process may be performed in advance (e.g., as part of a calibration process on the same or a corresponding device) to establish a correspondence between the number of oscillations and reaction progress. For example, a test run may be performed in which N oscillations are performed, after which the resulting contents of the chamber are analyzed, e.g., by performing a separation and using a detector to look for the presence or absence of a particular component, preferably the primary reactant. Multiple runs with different values ​​of N may be performed to establish a minimum value for N at which substantially no primary reactant is detected (meaning 100% of the primary reactant has reacted). The method can then be carried out on the actual sample with N (or more) oscillations if one wishes to react 100% of the primary components, or with a corresponding fraction of N oscillations if one wishes to allow the reaction to proceed to a lesser extent (e.g., 0.5 N if one wishes to react only 50% of the reactants).

[0067] In relation to running a reaction, the cycle time of the electric field sequence is selected depending on the particle mobility and electric field parameters, with the primary goal of focusing the particles into as small a volume as possible. In preferred embodiments, the cycle time is 0.1 to 100 seconds, preferably 0.1 to 10 seconds, and more preferably 1 to 5 seconds. Shorter cycle times are generally preferred because they result in narrower band oscillations, thereby keeping the material closer together for more of each cycle. However, if the particles have low mobility, the cycle time can be extended due to their relatively small momentum. Longer cycle times can be advantageous in relation to running a reaction because they help collect sample particles that may initially be widely distributed in the chamber, providing more time for the particles to align under the influence of the applied field profile. In some types of reactions, only small amounts of sample may be present, and therefore, it is important to collect as much sample as possible.

[0068] Preferably, in small-scale implementations of the reaction method (e.g., microfluidic environments), the first and second electric field gradients and the duration of each cycle are configured so that the charged particles oscillate along the major axis of the chamber, with an oscillation amplitude of less than 10 mm, preferably less than 3 mm, and more preferably less than 2 mm. Such small dimensions ensure that particles of each substance are sufficiently localized, thereby enhancing the reaction. Of course, in large-scale implementations where reactions are carried out, for example, to produce chemical products on an industrial scale, the oscillation amplitude may be substantially larger (and cycle times may need to be correspondingly longer).

[0069] Generally, when accelerating a reaction, the goal is to achieve maximum sample concentration and maximum band overlap during oscillation, which maximizes the reaction rate. Therefore, the use of small oscillation widths and steep electric field gradients, such as an electric field gradient varying from 0 to 800 V / cm over a first region with a width of 1 cm or less, is advantageous in this scenario.

[0070] As described above, the parameters of the electric field sequence and the respective mobilities of the charged particles of each material are preferably such that the respective bands of each material cross each other, preferably repeatedly, within the chamber during step b. For example, if a first band of high-mobility particles is moving in a first direction followed by a second band of lower-mobility particles, when the first electric field gradient is switched to a second electric field gradient, both bands begin to move in opposite directions. Due to its higher mobility, the first band overtakes the second band and, in doing so, crosses it. During the crossing, collisions occur between particles of each material.

[0071] In an advantageous development of the reaction method, after the electric field sequence has been performed a desired number of times (or after the reaction has reached a desired state), the outcome of the reaction may be investigated. In a preferred implementation, this may require separating the various particle types now present in the chamber, since some of the original unreacted material may remain. Therefore, the method preferably comprises, after step (ii), The method further includes separating the charged particles according to their type by performing electrophoresis on the contents of the reaction chamber, wherein the reaction products are separated from any remaining quantities of at least two substances. Since two or more reactions may occur in parallel, in the general case, all reaction products are preferably separated from each other (and from any remaining reactants). Exemplary electrophoresis processes suitable for this are described in more detail in connection with the third and fourth aspects of the invention.

[0072] Another additional advantage of the disclosed reaction method is that when used as a first step to a subsequent separation step (e.g., electrophoresis), lower concentrations of secondary reactants can be used (as is possible for the reasons explained above), reducing the chance that unreacted signal will overwhelm the small reaction product signal.

[0073] In this embodiment, the electrophoresis process may be carried out in a separation channel, which may or may not be fluidically connected to the chamber. For example, the separation channel may be separate from the chamber, and the chamber contents may be removed from the chamber and transferred to the separation chamber, e.g., by a pipette. Alternatively, the separation channel may be connected to the chamber via a suitable conduit or may be continuous with the channel (e.g., forming part of the same cavity). Separation may optionally be carried out simultaneously while another batch of reactants is reacted in the chamber.

[0074] In some implementations, alternatively or additionally, it may be desirable to output a product formed by the reaction, which may itself be a useful substance, for example, the product of an enzymatic reaction. Such reactions may be facilitated by the methods of the present invention. The product may be separated using the separation methods of the present invention, or alternative methods understood by those skilled in the art. Thus, the methods of the present invention preferably include, after step (ii) or after step (iii) (if performed), (iv) Preferably, the method further comprises extracting the reaction product by applying an electric field configured to move charged particles of the reaction product to a port through which the reaction product is removed.

[0075] In one scenario where the primary goal of the reaction is to produce a predetermined amount of reaction product, the method may be performed multiple times or continuously. For example, in the former case, a predetermined amount of each substance may be injected into the chamber, an electric field sequence applied to cause the reaction, and then the product extracted before the process begins again by injecting additional reactants. In the latter case, the electric field sequence may be repeated substantially continuously, with reactants injected at intervals and the contents extracted at predetermined times.

[0076] Thus, there is provided a method for detecting an analyte (viral and / or bacterial antigen) by means of a binding protein that specifically binds the analyte, said method comprising: (i) providing charged particles and binding proteins of an analyte; (ii) focusing charged particles in a chamber using the method of the first aspect of the invention, wherein the charged particles of the analyte and the binding protein form respective bands, the widths of the bands being successively reduced and the respective bands overlapping each other during at least a portion of the electric field sequence, thereby promoting interaction (such as reaction or binding) between the analyte and the binding protein.

[0077] The term "binding protein" refers to any protein capable of specifically binding to a target analyte (i.e., antigen). Preferably, the binding protein is an immunoglobulin molecule (i.e., antigen) or antigen-binding portion thereof that specifically binds to the target analyte (i.e., antigen). The terms "specifically bind," "specific binding," or "bind" are used interchangeably and refer to an immunoglobulin molecule, or antigen-binding portion thereof, that binds to an antigen or epitope of said antigen with greater affinity than it does to other antigens or epitopes. Such affinity is measured by the equilibrium dissociation constant (Kd) and can be measured using standard procedures known in the art. The antibody is preferably an IgG antibody or a fragment or derivative thereof, but may alternatively be an IgA, IgM, or IgE antibody or a fragment or derivative thereof. The binding protein may be a fragment, derivative, or analog of said antibody. The terms "fragment," "derivative," and "analog" refer to a polypeptide that substantially retains the same biological function or activity, e.g., the same antigen-binding specificity, of a full-sized immunoglobulin molecule. The immunoglobulin molecule may be, for example, a monoclonal antibody, a polyclonal antibody, a single heavy chain variable region, a Fab fragment, an scFV fragment, a diabody, or any other antibody fragment or fusion, as understood by those skilled in the art. Preferably, the immunoglobulin (antibody) is a monoclonal antibody.

[0078] Thus, the methods disclosed herein can be used to detect various viral and / or bacterial infections, including, but not limited to, human immunodeficiency virus (HIV), Ebola virus, hepatitis virus (A, B, or C), herpesvirus (e.g., VZV, HSV-I, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, coronavirus (e.g., SARS-CoV, SARS-CoV-2, MERS-CoV), respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, or arboviral encephalitis virus. Examples of possible bacterial infections include, but are not limited to, Chlamydia, Rickettsia, Mycobacteria, Staphylococcus, Streptococcus, Pneumococcus, Neisseria meningitidis and Neisseria gonorrhoeae, Klebsiella, Proteus, Serratia, Pseudomonas, Legionella, Corynebacterium diphtheriae, Salmonella, Bacillus, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus anthracis, Yersinia pestis, Mycobacterium leprosy, Mycobacterium lepromatosis, and Borrelia. Examples of possible parasitic infections that may be detected include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba lamblia, Cryptosporidium, Pneumocystis carinii, Plasmodium falciparum, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and Nippoststrongylus brasiliensis. Examples of possible fungal infections that may be detected include Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger), Mucor (Mucor, abscission, Rhizopus), Sporothrix schenckii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0079] A second aspect of the invention also provides a reactor for carrying out a reaction between at least two substances, the reactor comprising an apparatus for focusing charged particles according to the first aspect of the invention, wherein, in use, the charged particles of each substance form respective bands in the chamber, the widths of the bands being successively reduced and the respective bands overlapping each other during at least part of the electric field sequence, thereby facilitating the reaction between the substances.

[0080] The reactor achieves all of the benefits already described in relation to the second aspect, and the controller can be further programmed to control the electric field generator to carry out any of the preferred methods described above. The reactor can have any of the preferred features already described in relation to the apparatus of the first aspect of the invention.

[0081] In a preferred embodiment, the reactor further includes a separation channel fluidly connected to the chamber for performing electrophoresis therein, the electric field generator being further adapted to apply an electric field along the separation channel, and the controller being further configured to control the electric field generator to apply a particle separation electric field along the separation channel. As explained above, in some implementations it may be desirable to perform separation on the contents of the reaction chamber, and this is a convenient configuration for doing so.

[0082] Alternatively or in addition to an extraction port from the provided chamber, the reactor may further comprise an extraction port for extracting the separated band of particles from the separation channel. Typically, the extracted band will be the reaction product.

[0083] Preferably, the separation channel is connected to an outlet port of the chamber or the separation channel is continuous with the chamber and aligned along the major axis of the chamber, the benefits of which are explained in relation to the third aspect of the invention.

[0084] Advantageously, the reactor may further comprise a detector adapted to detect the separated bands of charged particles in the separation channel, the detector preferably being adapted to image the bands.

[0085] The third aspect of the invention utilizes the increased particle focusing achieved by the first aspect in the context of separating materials. As noted above, WO 2006 / 070176 discloses an electrophoretic technique for separating charged objects using a shifting field gradient. The objective of the disclosed method is for analyte molecules to achieve equilibrium between frictional forces exerted by the sieving medium on which the particles are supported and electric forces on the particles caused by the electric field. While the method is effective, it can be slow.

[0086] Accordingly, a third aspect of the present invention provides a method for separating at least two substances, comprising the steps of: (I) providing charged particles of at least two substances; (II) focusing charged particles in a chamber containing a sieving medium using the method of the first aspect of the invention, whereby the width of the group of charged particles is reduced; (III) performing electrophoresis on a group of charged particles along a separation channel containing a sieving medium, the separation channel being continuous with the chamber and aligned with a major axis of the chamber.

[0087] By focusing charged particles so that groups occupy a smaller volume (relative to the initially injected "plug") before separation, electrophoresis is completed more quickly than conventional methods. This is because similar particles are already closer to each other at the start of separation, and therefore, separate bands of similar particles emerge from the group of particles (i.e., each band can be distinguished from the next) than would otherwise be the case. Essentially, a narrower group of mixed particles necessarily results in narrower bands of similar particles, and these narrow bands are spaced apart more quickly than wider bands (because the trailing edge of a first wide band overlaps the leading edge of the next wide band for a longer period of time than would a narrower band). As a result, bands can be distinguished from each other (and thus materials separated) more quickly than previously possible. As a result, separation channels can be made correspondingly shorter than in conventional techniques, reducing the size of the device performing the separation. For example, this allows the use of short, straight, open-ended separation channels, as opposed to closed-loop channels with infinite length.

[0088] It will be appreciated that while the technique disclosed in WO 2006 / 070176 can achieve some degree of band focusing during separation, the primary advantage of this method is that it treats the focusing mechanism and band separation as a single, combined process. A shifting field profile propagates through the analyte molecules, causing them to move and ultimately achieve an equilibrium position in the field gradient. The separation and focusing processes occur at different rates, and there is a strong dependence of each on the characteristics of the electric field gradient. However, the dependence is very different for the focusing process compared to the separation process, and in WO 2006 / 070176, there must be a significant trade-off, since both must be achieved simultaneously by the same shifting electric field gradient. That is, the electric field gradient must be a compromise between optimizing band focusing and optimizing band separation.

[0089] Instead, the disclosed concept treats the concentration (focusing) and molecular separation processes separately and performs them in tandem. This allows the focusing and separation processes to be optimized separately in (at least) two different phases. Optimization of the focusing process is achieved by the aforementioned electric field sequence, which is preferably performed multiple times to oscillate the particles. The basic purpose of the electric field sequence in this phase is to avoid reaching an equilibrium condition. Only after the focusing process has reached a sufficient level, the disclosed method moves on to the separation step, which optimizes the separation while potentially maintaining a weaker focusing force.

[0090] Another benefit of separating the focusing and separation steps in this way relates to practical limitations on what electric field profile can be established with the available hardware. Typically, any particular hardware implementation is limited in terms of the maximum cumulative voltage it can safely handle and the maximum voltage limit its power modules can apply between two electrodes. For example, a device can be provided with 50 electrodes spaced 2.5 mm apart along a 12.5 cm long major axis. To generate an electric field of approximately 500 V / cm at a particular location along the channel, this requires a voltage difference of 125 V between adjacent electrodes at that location. If this level of electric field were applied along the entire length of the device, it would accumulate to 50 x 125 V = 6250 V across the 12.5 cm channel. This is an extremely high voltage and imposes problematic insulation requirements on the entire high-voltage circuit, including cables, high-voltage connectors, and insulation associated with fluids at the chip, fluid shorts due to spills, etc. However, in the approach disclosed herein, high electric fields can advantageously be applied initially only across a small portion of the device. For example, a steep focusing gradient may initially be applied across a first region encompassing approximately one-quarter of 12.5 cm, resulting in a cumulative voltage of only 1500 V. Thus, as described below, a gentler separation gradient (requiring a lower electric field, and therefore voltage) can be applied across a larger section of the device. This is significantly more manageable from a hardware perspective.

[0091] During the focusing phase of the method (step (II)), different particles may begin to form bands of similar particles, or may sort themselves into bands entirely. This is not necessary, but it is likely that at least some degree of separation will begin to occur as particles of different charge and / or mobility migrate differently through the sieving matrix under the influence of the electric field. During step (II), any bands formed will likely at least partially overlap one another and oscillate in the same region of space (defining the boundaries of the "group"), which narrows (as all bands do) as the method progresses. This is the same mechanism as already described in relation to the second aspect of the invention (however, in the third aspect, materials may or may not react with each other).

[0092] As explained in connection with the first aspect of the invention, the focusing method applied during step (II) utilizes a transition state of the force field as opposed to an equilibrium state. Indeed, while optimizing the focusing process, the equilibrium state is disturbed for at least n-1 analytes (bands) depending on the manner in which the electric field is applied. In other words, at most, as a special case, the equilibrium condition can be satisfied by only one band (not optimal, but not excluded as a possibility). All other bands (and usually all bands) are prevented from reaching the equilibrium condition in order to maximize focusing.

[0093] The separation channel is continuous with the chamber where focusing occurs and aligned with the chamber's major axis. This ensures that the dimension along which focusing occurs (parallel to the major axis) is the same as the dimension along which separation occurs. This is necessary to achieve the benefits described above. That is, increased localization along the same direction along which the bands are separated only helps to resolve the bands more quickly from each other. It should be noted that this does not mean that the separation channel needs to be straight. The vibration chamber can be located, for example, tangentially offshoot from a curved or circular separation channel. The chamber and separation channel preferably each form part of a continuous cavity, for example, formed in the surface of a substrate. Configurations such as those disclosed in WO 2006 / 070176 or WO 2012 / 153108 are suitable. The sieving matrix (usually the same in both the chamber and the separation channel) can be, for example, a fluid or a gel.

[0094] In step (II), the method of focusing the particles may employ any of the preferred features described in relation to the first aspect of the invention above.

[0095] The electric field sequence applied in step (II) is preferably repeated multiple times to achieve a greater degree of focusing before initiating separation in step (III). The number of cycles required depends on the particle mobility and the parameters of the electric field sequence, but in preferred embodiments, the sequence may be repeated at least 10 times, more preferably at least 30 times, and even more preferably at least 50 times. Note that these preferences are directed toward fewer repetitions than may be optimal when using the method to accelerate a reaction (in the second aspect of the invention), because in the context of separation, the primary objective is often speed and the rapid initiation of separation (to minimize overall run time) rather than maximizing the degree of focusing achieved. In contrast, when performing a reaction, it is primarily important to focus the particles as closely as possible to position them in close proximity to one another.

[0096] Alternatively or additionally, the electric field sequence may be repeated until the width of the group of charged particles reaches a predetermined threshold. For example, this may be monitored using a suitable detector capable of imaging the group of particles in the chamber. In a preferred embodiment, the predetermined threshold may be 10 mm or less, more preferably 1 mm or less, and even more preferably 0.1 mm or less. Again, these are values ​​suitable for small-scale implementations. Larger-scale versions are also possible. Depending on the implementation, width thresholds associated with individual bands may be employed rather than those associated with the group as a whole.

[0097] The cycle time of the electric field sequence is similarly selected to achieve a high number of oscillations within a short time frame, and in some preferred embodiments, the cycle time is 0.1 to 10 seconds, preferably 0.1 to 5 seconds, more preferably 1 to 3 seconds, and most preferably 1.5 to 2.5 seconds.

[0098] In a preferred implementation, the first and second electric field gradients and the duration of each cycle are configured so that the charged particles oscillate along the major axis of the chamber, with an amplitude of oscillation ranging from 0.1 to 10 mm, preferably 0.5 to 5 mm, and more preferably 1 to 3 mm. It will be appreciated that these values ​​will be larger than the exemplary group or band widths shown above, because the groups or bands typically do not occupy the entire space but move between the extremes of the oscillation amplitude.

[0099] The electrophoretic separation carried out in step (III) can be carried out using any available electrophoretic method, such as the application of a static uniform electric field along the channel. However, more preferably, in step (III), the electrophoretic method is III') applying a particle separation electric field along the separation channel, the particle separation electric field having a field profile that causes the charged particles to move relative to the sieving medium; III'') Varying the applied particle separation electric field to adjust the field profile for the separation channel, thereby separating the charged particles into respective bands of at least two materials under the combined influence of the electric forces due to the electric field and the hydrodynamic forces due to the sieving medium.

[0100] An example of such a process is disclosed in WO 2006 / 070176, the contents of which are incorporated herein by reference.

[0101] As noted above, the characteristics of the applied electric field are different in step (II) compared to step (III) to optimize it for performing various functions of particle focusing and subsequent band separation. In particular, the field profile of the particle separation electric field in step (III) preferably has an electric field gradient whose spatial rate of change of the electric field magnitude with distance along the separation channel is smaller than that of the first and second electric field gradients in step (II). In other words, the electric field gradient in step (III) has a smaller slope than that applied in step (II). This has the consequence that the equilibrium points of each respective band of similar particles (to which the particles migrate during step (III)) are separated by a greater distance from the next during separation. This allows the separated bands to be better resolved from each other. In contrast, a relatively steep gradient during the focusing phase (step (II)) is beneficial to better concentrate each band prior to separation. It should be noted that the decrease in slope between steps (II) and (III) does not result in the bands being dispersed (i.e., diffused). Rather, since there is still a slope in step (III), albeit a smaller one, the bands continue to narrow in step (III), albeit at a slower rate than in step (II). In other words, the flatter gradient used during the separation step does not result in dispersion.

[0102] It should be noted that the change in gradient from step (II) to step (III) can be discrete or gradual. In the latter case, the slope of the gradient can be progressively (stepwise or continuously) decreased, for example, before or during migration along the separation channel.

[0103] More generally, the two phases (i.e., steps (II) and (III)) may be somewhat merged, without a distinct point at which the focusing phase stops and the separation phase begins. For example, the electric field gradient applied during the final occurrence of step (b2) may be controlled to transition in a substantially continuous manner to the gradient used to perform the separation step (III).

[0104] It should be appreciated that the methods of the second and third aspects of the invention can be combined. That is, at least two substances injected into the chamber may react with each other during step (II) of the separation method, resulting in at least one reaction product in the manner described in connection with the second aspect of the invention. In this case, parameters of step (II), such as the electric field gradient slope, cycle time, and / or number of cycles, must be selected to balance the desire for a short overall separation run time against the desire to maximize reaction yield. Each of the at least two substances (reactants) may or may not remain present in the chamber (in reduced amounts) once the reaction occurs. Thus, the separation performed in step (III) may be on the contents of the chamber after the reaction, which will be different from the set of substances originally introduced. Thus, the substances separated into bands during step (III) may contain one or more substances different from those injected in step (I), or may consist exclusively of such different substances.

[0105] A third aspect of the invention further provides an electrophoresis device for separating charged particles, comprising an apparatus for focusing charged particles according to any of the first aspect of the invention and a separation channel continuous with the chamber and aligned along a major axis of the chamber, wherein the electric field generator is further adapted to apply an electric field along the separation channel, and the control device is further configured to control the electric field generator to apply the particle separation electric field along the separation channel.

[0106] The electrophoresis apparatus achieves all of the benefits described above in relation to the method of the third aspect. The apparatus for focusing particles may have any of the preferred features described in relation to the apparatus of the first aspect of the invention. The control device may be further programmed to control the electric field generator to perform any of the preferred features of the method of the third aspect mentioned above.

[0107] Preferably, the electrophoresis device further comprises an extraction port for extracting the separated bands of particles from the separation channel. If a reaction occurs between the originally injected substances, it will usually be desirable to extract the reaction products as mentioned in relation to the second aspect.

[0108] The electrophoresis apparatus may preferably further comprise a detector adapted to detect separated bands of charged particles in the separation channel, the detector preferably being adapted to image the bands.

[0109] In a third aspect of the invention, the electric field sequence disclosed in the first aspect of the invention is utilized to achieve strong focusing of the charged particles prior to separation. As noted above, reducing the width of the charged particle groups increases the speed of subsequent separation. This can also be achieved, albeit to a lesser extent, by alternative particle focusing techniques.

[0110] Accordingly, a fourth aspect of the present invention provides a method for separating at least two substances, comprising the steps of: (I) providing a sample containing at least two substances to provide charged particles of the at least two substances; (II) collecting charged particles in a chamber containing a sieving medium by applying a particle collection electric field gradient along at least a portion of a major axis of the chamber, wherein the charged particles move under the influence of the electric field gradient to form at least one group of charged particles, the leading edge of the group moving slower than the trailing edge of the group due to a smaller electric field magnitude acting on the particles at the leading edge, thereby reducing the width of the charged particles; and then (III) performing electrophoresis on a group of charged particles along a separation channel containing a sieving medium, the separation channel being continuous with the chamber and aligned with a major axis of the chamber.

[0111] In this method, step (II) does not require a sequence of applied electric field gradients. Rather, only one electric field gradient can be applied for the desired duration, with a profile designed to focus particles, before the applied electric field is changed to perform and optimize electrophoresis. This is a simpler approach than that of the third aspect of the invention, requires less sophisticated control means, and does not achieve as great a focusing effect. However, it also leads to an improvement in separation time, which may be sufficient for many applications.

[0112] The particle collection electric field gradient is preferably stationary during all or part of step (II) to collect particles over a wide area before separation begins. However, in alternative embodiments, the collection phase (step (II)) can overlap with the beginning of separation (step (III)), in which case the particle collection electric field gradient can be controlled to move along the major axis during all or at least the last part of step (II).

[0113] In some embodiments, the particle collection electric field gradient may include a zero-crossing point within a portion of the chamber's major axis, causing at least one group of charged particles to migrate toward the zero-crossing point. A zero-crossing point is a point in the electric field profile where the electric field magnitude is zero, with the electric field having a positive sign on one side of the point and a negative sign on the other side of the point. Charged particles on both sides experience forces that move them toward the zero-crossing point, creating a focusing effect. Alternatively, the particle collection electric field gradient may be all positive or all negative. For example, the particle collection electric field gradient may have the same form as the first electric field gradient or the second electric field gradient described in connection with the first embodiment.

[0114] The electrophoretic separation carried out in step (III) can be carried out using any available electrophoretic method, such as the application of a static uniform electric field along the channel. However, more preferably, in step (III), the electrophoretic method is III') applying a particle separation electric field along the separation channel, the particle separation electric field having a field profile that causes the charged particles to move relative to the sieving medium; III'') Varying the applied particle separation electric field to adjust the field profile for the separation channel, thereby separating the charged particles into respective bands of at least two materials under the combined influence of the electric forces due to the electric field and the hydrodynamic forces due to the sieving medium.

[0115] An example of such a process is disclosed in International Publication No. 2006 / 070176, the contents of which are incorporated herein by reference. It will be appreciated that the particle separation electric field has a profile different from that of the particle collection electric field gradient. Preferably, the field profile of the particle separation electric field in step (III) has an electric field gradient whose spatial rate of change of the electric field magnitude with distance along the separation channel is smaller than that of the particle collection electric field gradient in step (II). In other words, the particle separation electric field has a shallower slope than that of the particle collection electric field gradient. This has the consequence that the equilibrium points of each respective band of similar particles (to which the particles migrate during step (III)) are separated from the next by a greater distance during separation. This allows the separated bands to be better resolved from each other. In contrast, a relatively steep gradient is beneficial during the focusing phase (step (II)) to better concentrate each band prior to separation.

[0116] It should be noted that the change in gradient from step (II) to step (III) can be discrete or gradual. In the latter case, the slope of the gradient can be progressively (stepwise or continuously) decreased, for example, before or during migration along the separation channel. Thus, the electric field can be changed discretely or continuously from a particle collection electric field gradient to a particle separation electric field profile between steps (II) and (III), preferably while being moved from the chamber to the separation channel.

[0117] More generally, the two phases (i.e., steps (II) and (III)) may be somewhat merged, without a distinct point at which the focusing phase stops and the separation phase begins. For example, the electric field gradient applied during the final occurrence of step (b2) may be controlled to transition in a substantially continuous manner to the gradient used to perform the separation step (III).

[0118] It will be appreciated that although the substances injected into the chamber in step (I) may or may not react with each other, in this aspect of the invention the electric field applied in step (II) is not specifically designed to accelerate the reaction. Thus, the separation performed in step (III) may be on the post-reaction chamber contents which will be different from the set of substances originally introduced. Thus, the substances separated into bands during step (III) may include, or consist exclusively of, one or more substances different from those injected in step (I).

[0119] A fourth aspect of the invention is an electrophoresis device for separating charged particles of at least two substances, comprising: a chamber into which, in use, charged particles are injected into a sieving medium, the chamber having a major axis; a separation channel contiguous with the chamber and aligned with the major axis of the chamber; an electric field generator configured to apply an electric field along at least a first portion of a major axis of the chamber and along the separation channel; Also provided is an electrophoresis device comprising: a control device configured to control the electric field generator and programmed to control the electric field generator to first apply a particle collection electric field gradient along at least a portion of a major axis of the chamber, so that, in use, the charged particles move under the influence of the electric field gradient to form at least one group of charged particles, the leading edge of the group moving slower than the trailing edge of the group due to the smaller electric field magnitude acting on the particles at the leading edge, thereby reducing the width of the group of charged particles; and then apply a particle separation electric field along the separation channel, so that electrophoresis is performed on the group of charged particles.

[0120] The electrophoresis apparatus achieves all the benefits of the separation method described in the fourth aspect of the invention. Suitable examples of the physical configuration of the electrophoresis apparatus are disclosed in WO 2006 / 070176 or WO 2012 / 153108, although the control device would of course be programmed differently in the manner specified above. The control device may further be configured to control the electric field generator to carry out the method according to any of the preferred implementations described above.

[0121] The electric field generator can take any form that allows for the application of an electric field profile of the type described along the major axis of the chamber and separation channel. In a preferred embodiment, the electric field generator includes an array of electrodes arranged along at least a first portion of the major axis of the chamber and separation channel. In use, appropriate voltages are applied to each electrode to produce the desired electric field gradient. The electrode array is particularly well suited for generating electric field gradients in small-scale (e.g., microfluidic) implementations of the device. The electrode array can have the same general form as disclosed, for example, in WO 2006 / 070176 or WO 2012 / 153108.

[0122] Preferably, the chamber is provided with at least one input port for injecting a sample into the chamber, the sample comprising at least charged particles of at least two substances. Desirably, the separation channel is provided with at least one exit port for extraction of groups or bands of charged particles from the chamber. Additional electrodes may be associated with the input and / or exit ports to allow application of additional electric fields to inject particles into or extract particles from the channel.

[0123] In a preferred embodiment, the apparatus further comprises a detector adapted to detect groups or bands of charged particles in the separation channel, the detector preferably being adapted to image the groups or bands.

[0124] Illustrative examples of methods for focusing charged particles, reactants and / or separating materials, and corresponding apparatus therefor, will now be described with reference to the accompanying drawings. [Brief explanation of the drawings]

[0125] [Figure 1A] 1 shows a schematic diagram of an apparatus for focusing charged particles before performing a focusing method; [Figure 1B] 2 shows a schematic diagram of an apparatus for focusing charged particles after a focusing method has been performed; FIG. [Figure 2] FIG. 2 shows a schematic diagram of an exemplary chamber and electric field generating means that may form part of the apparatus shown in FIGS. 1A and 1B. [Figure 3A] 1 is a plot of electric field E versus distance x along the major axis of the chamber for two respective steps of a first embodiment of a focusing method according to an aspect of the present invention. [Figure 3B] 1 is a plot of electric field E versus distance x along the major axis of the chamber for two respective steps of a first embodiment of a focusing method according to an aspect of the present invention. [Figure 4A] 10 is a plot of electric field E versus distance x along the major axis of the chamber for two respective steps of a second embodiment of a focusing method according to an aspect of the invention. [Figure 4B] 10 is a plot of electric field E versus distance x along the major axis of the chamber for two respective steps of a second embodiment of a focusing method according to an aspect of the invention. [Figure 5] 1 is a flow chart illustrating steps performed in an embodiment of a focusing method according to an aspect of the present invention. [Figure 6A] 10 is a plot of the electric field E versus the distance x along the major axis of the chamber at three respective instants during a first step of a third embodiment of a focusing method according to an aspect of the present invention. [Figure 6B] 10 is a plot of the electric field E versus the distance x along the major axis of the chamber at three respective instants during a first step of a third embodiment of a focusing method according to an aspect of the present invention. [Figure 6C]10 is a plot of the electric field E versus the distance x along the major axis of the chamber at three respective instants during a first step of a third embodiment of a focusing method according to an aspect of the present invention. [Figure 7A] 10 is a plot of the electric field E versus the distance x along the major axis of the chamber at each instant during the second step of a third embodiment of a focusing method according to an aspect of the present invention. [Figure 7B] 10 is a plot of the electric field E versus the distance x along the major axis of the chamber at each instant during the second step of a third embodiment of a focusing method according to an aspect of the present invention. [Figure 7C] 10 is a plot of the electric field E versus the distance x along the major axis of the chamber at each instant during the second step of a third embodiment of a focusing method according to an aspect of the present invention. [Figure 8] 10 is a plot illustrating the results of a simulation modeling the behavior of an exemplary charged particle subjected to an embodiment of a focusing method according to an aspect of the present invention. [Figure 9] 10 is a plot illustrating the results of a simulation modeling the behavior of an exemplary charged particle subjected to an embodiment of a focusing method according to an aspect of the present invention. [Figure 10] 10 is a plot illustrating the results of a simulation modeling the behavior of an exemplary charged particle subjected to an embodiment of a focusing method according to an aspect of the present invention. [Figure 11] 10 is a plot illustrating the results of a simulation modeling the behavior of an exemplary charged particle subjected to an embodiment of a focusing method according to an aspect of the present invention. [Figure 12] 10 is a plot illustrating the results of a simulation modeling the behavior of an exemplary charged particle subjected to an embodiment of a focusing method according to an aspect of the present invention. [Figure 13] 10 is a flow chart illustrating steps performed in another embodiment of a focusing method in accordance with an aspect of the present invention. [Figure 14A] 1 is a plot of electric field E versus distance x along the major axis of the chamber, illustrating an exemplary first electric field gradient that may be applied during a first step of an electric field sequence. [Figure 14B]14A-14C are plots of electric field E versus distance x along the major axis of the chamber showing exemplary second electric field gradients that may be applied during the second step of the electric field sequence, each plot showing two respective electric field gradients appropriate for two respective stages of the method shown in FIG. [Figure 15] 10 is a plot illustrating the results of a simulation modeling the behavior of an exemplary charged particle subjected to another embodiment of a focusing method according to an aspect of the present invention. [Figure 16] 10 is a plot illustrating the results of a simulation modeling the behavior of an exemplary charged particle subjected to another embodiment of a focusing method according to an aspect of the present invention. [Figure 17] 10 is a plot illustrating the results of a simulation modeling the behavior of an exemplary charged particle subjected to another embodiment of a focusing method according to an aspect of the present invention. [Figure 18] 10 is a flow chart illustrating steps performed in a further embodiment of a focusing method according to an aspect of the present invention. [Figure 19] 19 is a plot of electric field E versus distance x along a major axis of the chamber showing an exemplary electrostatic field profile, an exemplary first electric field gradient, and an exemplary second electric field gradient, all suitable for use in the embodiment of FIG. 18. [Figure 20] FIG. 1 is a diagram illustrating an exemplary layout of device components suitable for use in an embodiment of the invention. [Figure 21] FIG. 1 is a diagram illustrating an exemplary layout of device components suitable for use in an embodiment of the invention. [Figure 22] 3 is a flow chart illustrating steps performed in an embodiment of a separation method according to an aspect of the present invention. [Figure 23] FIG. 23 is a schematic diagram illustrating an exemplary layout of apparatus components suitable for use in the method embodiment of FIG. 22. [Figure 24] FIG. 23 is a schematic diagram illustrating an exemplary layout of apparatus components suitable for use in the method embodiment of FIG. 22. [Figure 25A]23 is a plot of the electric field E versus the distance x along the major axis of the chamber during each step of an exemplary implementation of the method of FIG. 22. [Figure 25B] 23 is a plot of the electric field E versus the distance x along the major axis of the chamber during each step of an exemplary implementation of the method of FIG. 22. [Figure 25C] 23 is a plot of the electric field E versus the distance x along the major axis of the chamber during each step of an exemplary implementation of the method of FIG. 22. [Figure 26A] 1 is a plot showing the results of a simulation modeling the behavior of particles undergoing an exemplary separation method (i) according to a comparative example (dashed line) and (ii) according to an embodiment of the present invention (solid line). [Figure 26B] 1 is a plot showing the results of a simulation modeling the behavior of particles undergoing an exemplary separation method (i) according to a comparative example (dashed line) and (ii) according to an embodiment of the present invention (solid line). [Figure 27A] 1 is a plot showing the results of a simulation modeling the behavior of particles undergoing an exemplary separation method (i) according to a comparative example (dashed line) and (ii) according to an embodiment of the present invention (solid line). [Figure 27B] 1A-1C are plots showing the results of a simulation modeling the behavior of particles undergoing an exemplary separation method (i) according to a comparative example (dashed line) and (ii) according to an embodiment of the present invention (solid line). 1B-1C are plots showing the results of a simulation modeling the behavior of particles undergoing an exemplary separation method (i) according to a comparative example (dashed line) and (ii) according to an embodiment of the present invention (solid line). [Figure 28A] 10 is a plot of electric field E versus distance x along the major axis of the chamber during each step of another embodiment of a separation method according to an embodiment of the present invention. [Figure 28B] 10 is a plot of electric field E versus distance x along the major axis of the chamber during each step of another embodiment of a separation method according to an embodiment of the present invention. [Figure 28C] 10 is a plot of electric field E versus distance x along the major axis of the chamber during each step of another embodiment of a separation method according to an embodiment of the present invention. [Figure 29] 10 is a plot of the electric field E versus distance x along the major axis of the chamber in one step of a first embodiment of the focusing method, showing the effect on particles of mixed charge signs. [Figure 30A] 10 is a plot of electric field E versus distance x along the major axis of the chamber for two respective steps of another embodiment of a focusing method according to an embodiment of the present invention. [Figure 30B] 10 is a plot of electric field E versus distance x along the major axis of the chamber for two respective steps of another embodiment of a focusing method according to an embodiment of the present invention. [Figure 31A] 10 is a plot of electric field E versus distance x along the major axis of the chamber for two respective steps of a further example of a focusing method according to an embodiment of the invention. [Figure 31B] 10 is a plot of electric field E versus distance x along the major axis of the chamber for two respective steps of a further example of a focusing method according to an embodiment of the invention. [Figure 32A] 1 is a plot showing test results for Covid-19 performed using a method according to an embodiment of the invention utilizing a vibration stage, showing a negative result. [Figure 32B] FIG. 32B(i) is a plot showing the results of a test for Covid-19 performed using a method according to an embodiment of the invention utilizing a vibration stage, showing a positive result; FIG. 32B(i) shows an enlarged detail of the plot of FIG. 32B. [Figure 33] 1 is a plot showing the results of a (positive) test for Covid-19 performed using a method according to an embodiment of the invention that utilizes a static collection stage. [Figure 34A] 1 is a plot showing the results of a test for Lassa virus performed using a method according to an embodiment of the invention utilizing a vibration stage, showing a negative result. [Figure 34B] 1 is a plot showing the results of a test for Lassa virus performed using a method according to an embodiment of the invention utilizing a vibration stage, showing a positive result. [Figure 35]10 is a plot showing the results of a (positive) test for influenza B performed using a method according to an embodiment of the invention utilizing a static collection stage. DETAILED DESCRIPTION OF THE INVENTION

[0126] The following description focuses on small-scale, e.g., microfluidic, implementations of the invention. As explained in more detail below, the methods described herein are particularly useful in conjunction with biological assays. In particular, the methods disclosed herein are particularly useful for determining binding between two substances (e.g., protein-protein interactions or protein-DNA interactions) and / or characterizing said substances. Accordingly, the present invention can be used as an alternative to many commonly used biological assays in this field, such as immunoprecipitation assays, pull-down assays, mobility shift assays, ELISA assays, and Western blots. The advantages of the present invention are numerous in this regard, including, but not limited to, reduced assay run time and improved interaction between two substances. In instances where the substances are proteins, a further advantage is that the method allows binding of proteins in their undenatured state to be assessed and characterized. This is in contrast to other commonly used methods in this field, such as Western blots and SDS-PAGE, which require protein denaturation. Traditional denaturing electrophoresis methods do not offer the advantages provided by the present invention. Furthermore, the speed and precision of the methods of the invention may allow for the detection of fleeting interactions between molecules, which may be useful for identifying receptor ligands in heterogeneous mixtures of analytes. The methods may also be useful for detecting low-affinity interactions between proteins. However, as noted above, the same principles can be applied on a significantly larger scale if desired. In such cases, implementations of the invention may be used in applications such as producing chemical or biochemical substances. For example, reaction products may be produced using the methods of the invention and then separated from the enzyme and substance.

[0127] Another example of a potential use of the methods of the present invention is the purification of analytes, such as proteins, using binding molecules such as antibodies. Similar to a typical immunoprecipitation (pull-down) assay, a sample solution containing the analyte can be incubated with an antibody specific for the analyte of interest and subjected to the methods of the present invention. This concentrates the analyte at a predetermined location in the field. A separation phase can then be used to separate the analyte from the antibody and then extract it. The non-denaturing conditions of the method help produce natively folded proteins that can be used as needed. Of course, the analyte can be tagged using methods known to those skilled in the art, and a binding molecule can be used to isolate the analyte by interaction with the tag. Thus, the present invention may provide an improved alternative method for identifying protein-protein interactions over, for example, the method described in Arifuzzaman et al. (2006) Large-scale identification of protein-protein interaction of Escherichia coli K-12, Genome Res. 16(5):686-91 (incorporated herein by reference).

[0128] The present invention can be used in conjunction with other product preparation methods in synthetic biology processes to accelerate interactions between key molecules of interest at critical times in the production process. For example, the methods of the present invention can be used in parallel with cell-free protein synthesis methods, such as those described in Khambhati et al. (2019) Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems, Frontiers in Bioengineering and Biotechnology 7 (incorporated herein by reference) and / or Gregorio et al. (2019) A User's Guide to Cell-Free Protein Synthesis, Methods Protoc. 2(1):24 (incorporated herein by reference). The methods of the present invention can play a key role in accelerating the synthetic biology workflow at multiple levels throughout the algorithm, from DNA synthesis to protein expression and purification, and in identifying and characterizing protein-protein interactions, all with high speed and accuracy.

[0129] 1A and 1B schematically illustrate an apparatus 1 suitable for use in embodiments of the invention. The apparatus 1 includes a chamber 2 that defines a volume in which, in use, charged particles 10 are placed. For example, the chamber 2 may be formed as a cavity (or part of a larger cavity) in the surface of a substrate. The charged particles 10 may be in a fluid 8, such as a solution having a predetermined pH to control the apparent charge of the particles 10, or a sieving matrix. In some cases, the fluid 8 can perform multiple functions, such as charge control and sieving. The fluid 8 can be replaced by a gel or other sieving matrix. In other cases, the fluid may not be present, for example, in a gaseous implementation. It should be appreciated that a sieving matrix need not be present for particle focusing to occur in the manner to be described, since the particles 10 are not required to be subjected to hydrodynamic forces (although, if a fluid or other sieving matrix is ​​present, the particles 10 would, of course).

[0130] Chamber 2 has a major axis x along which an electric field can be established by electric field generator 4. It should be noted that while in FIGS. 1A and 1B the major axis is shown parallel to the elongated direction of the chamber, this is not required, and in fact chamber 2 need not be elongated. In this embodiment, electric field generator 4 includes an array of electrodes spaced apart along major axis x. The array includes at least two electrodes, and preferably three or more (most preferably significantly more). It will be appreciated that the geometry of the chamber may also affect the electric field profile; for example, if the cross-sectional area of ​​the chamber is not constant along the major axis, this may contribute to establishing the desired electric field spacing. Examples of suitable structures are provided below.

[0131] The electrodes are connected to a suitable voltage source (not shown) via a controller 6. The controller is configured to control the voltage level applied to each electrode so as to apply a desired electric field profile inside chamber 2 along major axis x. That is, electric field generator 4 can control both the magnitude and sign of the electric field at each point within chamber 2 along major axis x (at least along a portion of major axis x). Examples of suitable electric field generators, including electrode arrangement configurations and controllers, are disclosed in WO 2006 / 070176 and WO 2012 / 153108, both of which are incorporated herein by reference. The portions of the separation channel disclosed in these documents are also suitable for use as chamber 2 in the methods of the present invention.

[0132] The particles 10 to be focused are injected into the chamber 2, for example, by a pipette or through an input port (not shown) equipped with one or more electrodes for applying an appropriate force to move the particles into the chamber. In this example, all of the charged particles 10 have the same apparent charge sign (e.g., negative), but the charge signs may be of different types and therefore may have different negative charge levels. The particles 10 form groups 12 that initially disperse across a wide volume of the chamber along the major axis x, as shown in FIG. 1A. After performing the focusing method described below, the width of the group of particles 12 is reduced, as shown in FIG. 1B. That is, the group 12 occupies a smaller distance along the major axis x than before. This process therefore spatially concentrates the group of particles into a smaller volume. It should be noted that the concentrated group of particles 12 may not include all of the particles 10 originally injected into the chamber 2. There may be some outliers that remain uncaptured and unfocused by this process. However, the method is preferably configured so that the majority of particles 10 are trapped and focused in groups, and methods for enhancing this are described below.

[0133] FIG. 2 again shows chamber 2 and identifies a first portion P1 of the principal axis, within which the electric field sequence is applied during the focusing process. This first portion P1 can extend across the entire size of chamber 2, but more commonly, is a portion thereof. First portion P1 is a region along the principal axis to which an electric field gradient is applied at a point during the electric field sequence. A first region R1 of the principal axis is also defined, substantially fixed relative to the principal axis. Particles positioned within first region R1 of the appropriate electrical field code are focused by this process. In the first embodiment described with reference to FIGS. 3A and 3B, the extent of first region R1 is equal to the extent of first portion P1, but this need not be the case.

[0134] The focusing method requires the application of an electric field sequence along a first portion P1 of the major axis x. A first example of a suitable electric field sequence used in the first embodiment is described with reference to FIGS. 3A and 3B. In a first step S111 of the sequence, a first electric field gradient 21 is applied via an electric field generator 4 throughout a first region R1. The first electric field gradient 21 includes positive values ​​of the electric field E everywhere in the first region R1, and the magnitude of the electric field decreases in a first direction (here, the +x direction) along the major axis. The effect of the first electric field gradient 21 on charged particles is illustrated with reference to just two exemplary particles 10a and 10b located at opposite edges of a particle group 12. The group 12 of charged particles moves in a first direction along the major axis under the influence of the first electric field gradient 21. However, the leading edge of group 12 (represented by particle 10b) moves slower than the trailing edge of the group (represented by particle 10a) due to the smaller electric field magnitude acting on the particles at the leading edge. This results in an electric field profile shape that decreases in value in the +x direction, and therefore particles further along the major axis experience less force than those behind them (as indicated by the different sized arrows representing the electric force on each particle in the diagram). As a result, the trailing edge "catches up" to a certain extent to the leading edge, reducing the width of the group of charged particles 12.

[0135] In the second step S112 of the electric field sequence, a second electric field gradient 22 is applied across the entire first region R1 (instead of the first electric field application). This is shown in FIG. 3B. The second electric field gradient 22 includes a negative value of the electric field E everywhere in the first region R1, and the magnitude of the electric field decreases along the major axis in a second direction opposite the first direction (here, the -x direction). As a result, particles 10a and 10b flip and now move in the second direction. Again, the leading edge of group 12 (here represented by particle 10a) experiences a smaller force than the trailing edge of group 12 (here represented by particle 10b). This causes particle 10b to move faster than particle 10a, again narrowing the width of the group.

[0136] Essentially, what happens is that when the applied electric field gradient pushes the group "to the right" (FIG. 3A), the left side of the group 12 experiences a higher E field than the right side of the group. Therefore, the left side moves faster than the right side, resulting in focusing. Then, when the electric field gradient is reversed (FIG. 3B), the electric field gradient pushes the particles toward the left, and the same thing happens in reverse (still focusing). This mechanism does not require hydrodynamic forces or sieving. The steeper the slope of the electric field gradients 21, 22, the greater the focusing effect. The difference in electric force between the front (leading edge) and back (trailing edge) of the band is proportional to: dE~E leading -E trailing (1) Here, E represents the electric field, and therefore the focusing speed is proportional to the gradient of the electric field.

[0137] The magnitude of the applied electric field also has an effect, with larger field magnitudes causing the particles to stay away from their equilibrium condition, which also improves the focusing effect. To understand the mechanism behind this, we need to consider a more complete set of forces acting on each particle. F leading =qE leading -fv leading (2) F trailing =qE trailing -fvtrailing (3)

[0138] where q is the charge on the particle, f is the friction coefficient, and v is the velocity. leading is the force on the representative particle at the leading edge of the group, and F trailing is the force on a representative particle at the trailing edge. This model accounts for electrophoretic conditions, where molecular mobility (resistance) becomes significant.

[0139] Therefore, F leading -F trailing The difference between f(v leading -v trailing ). However, the latter term is a function of the edge velocity, and therefore the equation is nonlinear. Indeed, for an electric field profile moving with velocity k, equations (2) and (3) vanish when the particle is at equilibrium in the electric field profile (friction equals the electric force). In contrast, the force is greatest away from equilibrium, where the term qE dominates fv. This means that the force is proportional to the distance from the equilibrium position and the focusing force (which is effectively F leading -F trailing This difference is approximately q(E front -E back ) and is zero at equilibrium.

[0140] In the static field case, the equilibrium point is at E = 0. Thus, in the static field case, a sufficiently non-zero average electric field magnitude experienced by the bands is the condition for moving away from the equilibrium state and thus accelerating focusing.

[0141] A similar argument applies to non-zero field velocities. The further the particle moves away from E=0, the greater the focusing force the band experiences. It should be noted that in this case, if the average particle position is far enough away from the equilibrium point for a particular field velocity, especially on the higher side of the equilibrium point, the field velocity can actually be considered zero. For example, if the equilibrium field is 100 V / cm for a field moving at 0.5 mm / s, but the band's current average position is 500 V / cm, then the field velocity is irrelevant, since the focusing velocity will not be different for field velocities of 0.5 mm / s and 0 mm / s as long as the band remains this far from equilibrium. In the example shown above, the first electric field gradient 21 is positive and the second electric field gradient 22 is negative, but it will be appreciated that the reverse is also possible. Also, the slopes of the two gradients 21, 22 can be reversed (i.e., the first direction can be in the -x direction and the second direction can be in the +x direction), in which case the process will be effective for particles of opposite charge signs, as will be further explained in relation to Figures 29-31.

[0142] Steps S111 and S112 together form one cycle of the electric field sequence. In some embodiments, this cycle can be performed one time. More preferably, however, the sequence is repeated multiple times, potentially tens or even hundreds of times. By repeating the sequence, the group of particles 12 oscillates along the major axis of the chamber about an average position, with one oscillation occurring for each repeat cycle. The more repeat cycles performed (and thus the greater the number of oscillations), the greater the degree of focusing ultimately achieved. As explained below, the actual number of repetitions performed will depend on the desired results, which may vary depending on the application.

[0143] In the illustrated example, the slope of the first electric field gradient 21 (i.e., the rate of change of the electric field E over the distance x) is the same as that of the second electric field gradient 22. This is preferred to achieve a more symmetrical result (the degree of narrowing and the amount of movement of the group 12 are the same in both steps), but this is not required. For the same reason, the maximum and minimum magnitudes of the electric field gradients 21, 22 are preferably substantially the same (as shown). It should also be noted that while the exemplary electric field gradients 21, 22 are linear, this is also not required; in other cases the electric field gradients can be curved (e.g., parabolic or exponential). Preferably, the two electric field gradients 21, 22 have the same shape as each other. It should also be noted that whether the electric field profile in steps S111 and / or S112 lies outside the first region R1 (and if so, what characteristics it may have) is not important and does not affect the operation of the described method. Thus, although Figures 3A and 3B show zero electric field outside the first region R1, this may or may not be the case in practice.

[0144] A second example, in which the electric field is also applied outside the first region R1, is now described with reference to FIGS. 4A and 4B. FIGS. 4A and 4B show the electric field profiles applied along the major axis x of chamber 2 during the first (S111) and second (S112) steps of the electric field sequence, respectively. In this case, a compound electric field gradient 29 is used, including both a first portion constituting first electric field gradient 21 and a second portion constituting second electric field gradient 22. As in the previous example, first electric field gradient 21 is all positive and decreases in the +x direction, while second electric field gradient 22 is all negative and decreases in the -x direction. In this example, compound electric field gradient 29 is continuous, and the two portions intersect at zero-crossing point 28, together forming a zero-crossing linear electric field gradient. However, in other cases, the composite electric field gradient 29 may be discontinuous, for example having a region of zero field magnitude between the two portions 21, 22.

[0145] The same compound electric field gradient 29 is applied to the chamber in both the first and second steps of the sequence, but at different positions along the major axis x. Thus, as shown in FIG. 4A , in the first step S111 of the sequence, the compound electric field gradient 29 is applied such that a first portion thereof, corresponding to the first electric field gradient 21, coincides with the first region R1. In the second step S112 of the sequence, the compound electric field gradient 29 is applied at a position shifted along the major axis x such that a second portion thereof, corresponding to the second electric field gradient 22, coincides with the first region R1. Here, the positions of the particle groups are represented by line 12, which can be seen to narrow as the method progresses in the same manner as previously described.

[0146] FIG. 5 is a flowchart illustrating steps in an exemplary focusing method employing the principles already described. Steps indicated by dashed lines are optional. In a first step S111, a first electric field gradient 21 is applied in a first region R1, which may take the form shown in FIG. 3A or 4A, for example. Then, in step S112, the electric field is switched to a second electric field gradient 22, as shown in FIG. 3B or 4B. This switching may be discrete, with no intermediate transition from one gradient to the other, and may preferably be instantaneous (i.e., substantially no time elapses between steps S111 and S112). In other words, the applied electric field in the first region R1 "jumps" from the first electric field gradient to the second electric field gradient, and vice versa. However, in other implementations, there may be one or more other steps performed between steps S111 and S112, such as a transition step S111′, during which the first electric field gradient is changed to a second electric field gradient. For example, if the two electric field gradients are implemented as part of a compound electric field gradient 29 (as in FIGS. 4A and 4B ), this may include translating the compound electric field gradient 29 along the major axis x during transition step S111′. Similarly, step S112 may be followed by a transition step S112′, during which the second electric field gradient is changed to the first electric field gradient, preparing for the sequence to be repeated. Again, this may include translating the compound electric field gradient 29 back to its original position. Thus, in method embodiments, the process may include repeatedly moving the compound electric field gradient 29 back and forth along the axis x.

[0147] If transition steps S111′ and / or S112′ are employed, each is preferably shorter in duration than either of steps S111 and S112. The transition steps are ideally as short as possible so that even highly mobile particles do not have enough time to reach a position in the electric field profile with a low or zero electric field magnitude (which may reside in the first region during the transition), which could impair the focusing effect. Ensuring that particles experience a relatively high electric field magnitude throughout as much of the process as possible enhances the focusing achieved. As seen by the simulations below, the method of the present disclosure has the result that particles “see” a non-zero average electric field magnitude (averaged over the duration of the electric field sequence). This is true whether or not transition steps S111′ and / or S112′ are included. In a preferred embodiment, the duration of each of the transition steps S111' and S112' (if provided) is at least 10 times shorter than the duration of steps S111 and S112, more preferably at least 50 times shorter, and most preferably at least 100 times shorter.

[0148] Thus, steps S111 and S112 preferably together form a large proportion of the electric field sequence. In some preferred embodiments, steps S111 and S112 together account for at least 80%, preferably at least 90%, and more preferably at least 99% of the duration of each cycle. The overall duration of the sequence (i.e., the cycle repetition time) significantly impacts the results achieved and is selected depending on the application and desired results. Preferably, the duration of each step S111 and S112 is substantially the same. The longer the sequence duration, the more time the group of particles 12 has to move in each step before reversing. This leads to greater particle movement and wider oscillation of the group 12 back and forth along the major axis x. Depending on the application, this may or may not be desirable, as explained below.

[0149] In general, it has been found that electric field sequences having a total duration (cycle time) of 0.1 to 100 seconds, preferably 0.1 to 10 seconds, more preferably 0.1 to 5 seconds, and most preferably 1 to 3 seconds, produce good results. For example, in one example (described in further detail below), the duration of each of steps S111 and S112 was 1.15 seconds, with instantaneous transitions between them, forming a cycle time of 2.3 seconds, corresponding to a single oscillation of group 12.

[0150] Repeating the electric field sequence causes the particles to oscillate along their major axes around a mean position within the chamber. When multiple different particle types are present, they form bands, all of which oscillate around the same mean position but with different amplitudes depending on their molecular mobility or mass. The resulting width of the oscillations exhibited by each group (or band) of particles depends on the particle's mobility and the characteristics of the applied electric field sequence. Each band oscillates and focuses.

[0151] As noted above, the desired oscillation amplitude will vary depending on the application, but in some general cases the first and second electric field gradients and the duration of each cycle may be configured to provide an oscillation amplitude of 0.1-10 mm, preferably 0.5-5 mm, and more preferably 1-3 mm. The oscillation amplitude may preferably be narrowed as the method progresses, for example, by decreasing the cycle time as described further below.

[0152] The electric field sequence (consisting of steps S111 and S112, and optionally steps S111′ and S112′ in this example) is executed one or more times until sufficient focusing is determined to be achieved in step S113. This may mean performing a certain number of repetitions (e.g., determined by modeling to provide sufficient focusing) or may include using a detector to monitor the width of group 12 (e.g., imaging the contents of chamber 2) and identifying when the width reaches a predetermined threshold. At that stage, the electric field sequence may be stopped and the focusing process terminated (S114), although there may be further steps, such as performing separation on the contents of the chamber and / or extracting one or more substances, as described below. In some embodiments, achieving a desired degree of focusing may not trigger the end of the electric field sequence repetitions, as it may be desirable to continue oscillation for a predetermined period of time (or indefinitely) to maintain the particle group in a focused state and avoid it spreading as a result of diffusion.

[0153] As alluded to above, it is desirable to keep the average electric field magnitude experienced by the particles high during the electric field sequence in order to maintain a higher electric force on each particle and a greater focusing effect. One embodiment in which this is enhanced by further refinement of the electric field sequence is now described with reference to FIGS. 6 and 7. Here, the shapes of the first and second electric field gradients 21, 22 applied during the first and second steps S111 and S112, respectively, are the same as those described in connection with the first embodiment (FIGS. 3A and 3B). In the first embodiment, as the group of particles 10 moves in the +x direction during step S111, the group necessarily moves toward regions of lower electric field magnitude (due to the slope of gradient 21), thereby lowering the overall average electric field seen by the particles. Similarly, the same occurs in step S112. This embodiment improves on this by also translating the first and second electric field gradients 21, 22 during each step S111 and S112, respectively.

[0154] 6A, 6B, and 6C illustrate this translation of the first electric field gradient 21 during step S111. At a first instant in time, t1, the electric field gradient 21 is positioned at a first position, as shown in FIG. 6A. At the location of the group of particles 12, the electric field has a magnitude of approximately (+)E1 (the exact value being greater at the trailing edge of the group than at the leading edge). At a later time t2 (still within step S111), as shown in FIG. 6B, the particles 12 are moving in the +x direction but have (under the control of the controller 6) an electric field gradient 21 such that the particles are still subjected to an electric field magnitude close to E1. The same is true at an even later time t3, as shown in FIG. 6C. Thus, the average electric field magnitude seen by the particles remains higher throughout step S111 than if the electric field gradient 21 were static between steps.

[0155] 7A, 7B, and 7C show the same principle applied in a second step S112, where the second electric field gradient 22 is translated in the −x direction as the steps proceed, maintaining the magnitude of the electric field seen by the particle 10 near a value (−)E2 that is larger than if the electric field gradient were static.

[0156] It will be appreciated that although this concept is described as if the electric field experienced by particle 10 remains substantially constant during each respective step (the electric field gradients 21, 22 are translated at approximately the same rate as the particle is moving), this is not likely to be the case in practice. However, there is still some increase in the average electric field seen relative to a static scenario. It should also be noted that although this improvement is described as a variation on the first embodiment, it is equally applicable to the second embodiment and all of the embodiments described below.

[0157] The results of computer simulations performed to demonstrate the effects achieved by the methods disclosed herein are now described with reference to Figures 8-12. Here, the behavior of a mixed set of six particle types, each of different mobility but of the same charge sign (i.e., all positive or all negative), was modeled in the presence of an electric field sequence according to an embodiment of the present invention. As can be seen, each particle type separated into distinct groups or bands of similar particles with the same mobility during the process. The mobilities of each group are shown in Table 1 below. [Table 1]

[0158] The sequentially applied first and second electric field gradients 21, 22 were formed from a compound electric field gradient of a shape similar to that shown in Figures 4A and 4B. No transition steps were employed, so switching between gradients was essentially instantaneous. In this case, the first cycle formed a selective "collection" phase (described below with reference to Figures 13 and 14), during which the applied electric field gradient was relatively shallow. The compound electric field gradient extended from +800 V / cm to -800 V / cm across 48 nodes (121.92 mm), each node corresponding to one electrode in the array. Each step had a duration of 1.25 seconds, during which the electric field gradient was selectively moved in the relevant direction at a slow speed of 0.015 cm / s (as described above with reference to Figures 6 and 7). After this "collection" cycle was completed, the electric field parameters were changed to perform a "focusing" phase. Here, the applied electric field gradient was steeper, formed by a compound electric field gradient extending from +800 V / cm to -800 V / cm across seven nodes (17.78 mm). In each repetition, the first electric field gradient (step S111) was applied for 1.25 seconds, followed by the second electric field gradient (step S112) for 1.25 seconds, forming a cycle time of 2.5 seconds. During each step, each electric field gradient was translated along its major axis at 0.03 cm / s (optional) in the manner described above with reference to Figures 6 and 7. During the focusing phase, the electric field sequence was repeated a total of 30 times to achieve the desired degree of focusing. This was followed by a separation step beginning at approximately t = 77.5 s. For the moment, only the focusing phase will be described, and the separation phase will be reversed.

[0159] Figures 8, 9, and 10 are plots showing three different views of the trajectories of six bands of particles during a run. Some of the six paths overlap each other, at least in certain places, which is why fewer than six traces are visible in some cases. Figure 8 is a space-time display, showing the band's position along the major axis (i.e., x-axis position) on the vertical axis and time on the horizontal axis. The oscillatory motion of the bands, centered around an average position of approximately 2 cm along the major axis, can clearly be seen. Although the six trajectories initially have different average positions, they eventually synchronize with the switching electric field gradient and oscillate around a common center. It can also be seen that the overlap of the various bands increases as the oscillation progresses, indicating that the overall width of the group of particles (i.e., the set of bands) is also reduced over time.

[0160] Figure 9 shows the time-charged field magnitude seen by each group (i.e., the electric field experienced by each group at each point in time - note also that this is not the same as the applied electric field, as each group takes time to "catch up" to the applied field - the sign of the electric field is not taken into account). It can be seen that the average electric field magnitude is about 245 V / cm. Clearly, this value is not zero, which indicates a strong focusing effect of the electric field sequence.

[0161] FIG. 10 shows position along the major axis x on the horizontal axis versus the electric field on the vertical axis. Because the horizontal axis is position x rather than time, the lines can be traced "backward" and forward. The plot shows that all six trajectories initially follow larger closed loops and gradually converge to a narrower loop with an average electric field value (magnitude) of approximately 245 V / cm. The "drift" exhibited by the trajectories may be caused by the initial position of the injected "plug" of particles not being aligned with the center of the first region across which the converging electric field sequence is applied; as the method progresses, the particles move toward that center. FIG. 11 shows an enlarged version of a portion of one of the trajectories in FIG. 10. Here, the closed-loop trajectories of one group of particles can be clearly seen. The first part of the trace (arrow (i)) shows particles moving in the +x direction during step S111 and experiencing the decrease in electric field strength (due to the ramping of the gradient, as explained above) seen as they do so from approximately +260 V / cm to approximately +235 V / cm. Path (ii) shows the electric field switching to step S112, at which point the particle stops moving and suddenly experiences a high electric field in the opposite direction. Path (iii) shows the particle moving in the x-direction during step S112, and again the electric field magnitude decreases from about -260 V / cm to about -235 V / cm. The electric field direction then switches again, as shown by line (iv). For most of the cycle time, the particle experiences either portion (i) or portion (iii) of the loop shown. Portions (ii) and (iv) have zero or near-zero duration. Thus, the average electric field magnitude "see" by the particle over the entire cycle is largely determined by that occurring during portions (i) and (iii), which is then about 245 V / cm.

[0162] Figure 12 is a plot showing the relationship between cycle time (i.e., the total duration of one instant of the electric field sequence) and oscillation amplitude, and its dependence on particle mobility. The exemplary particles used in the simulations here have the following respective mobilities: high mobility ("fast" trace = 2.5 x 10 -3(m / s) / (V / m), intermediate mobility ("middle" trace) = 1.25 × 10 -4 (m / s) / (V / m), and low mobility ("slow" trace) = 2.5 x 10 -5 (m / s) (V / m). To achieve an oscillation amplitude of 0.5 mm to 5 mm (effective for many applications), the required oscillation duration can be found to vary from 0.2 s to over 100 s. The longer the cycle time, the greater the amplitude of the oscillation induced by the electric field sequence. Particles with higher mobility have larger oscillation amplitudes than particles with lower mobility. This is because higher mobility particles can move a greater distance during each step of the cycle. When oscillating, it is often desirable to minimize the oscillation amplitude, since a smaller oscillation amplitude usually reduces focusing time and also reduces the size of the device.

[0163] Particle oscillations may tend to narrow over time for the same electric field gradient. However, as they narrow, the particles remain in regions of progressively lower electric field magnitude, which is undesirable. To reduce this effect, in some preferred embodiments, the oscillation width may be narrowed as the method progresses, for example, by reducing the cycle time. This helps reduce or eliminate this "drop" of particles to lower electric field values. Essentially, this has a similar effect to the "translation" of the first and second electric field gradients 21, 22 performed in preferred embodiments as described above in connection with FIGS. 6 and 7. Both of these approaches can be applied in combination if desired.

[0164] As mentioned at the beginning, the initial sample (or "plug") of charged particles can be spread over a large volume of chamber 2, its length can be several centimeters. In such a case, if a method of the kind described above is applied over a first region R1 that is only a few millimeters wide, the injected plug can be "cropped," leaving a significant proportion of the charged particles outside the applied electric field sequence, which do not form part of the group of particles to be focused. To solve this, embodiments of the invention can include a particle collection step before the focusing step begins.

[0165] A first example of such an embodiment is described with reference to FIGS. 13 and 14. In this case, the particle collection phase and the subsequent particle focusing phase each require the application of an electric field sequence of the type already described, but with different characteristics. The two phases can be separate, or one can merge into the other by gradually changing the sequence parameters. FIG. 13 is a flowchart illustrating an exemplary technique. Here, steps S101, S102, and S103 form the particle collection phase, while steps S111, S112, and S113 form the focusing phase. In the particle collection phase (S1), first and second electric field gradients are applied over a relatively wide first region R1 (S1). This is shown in FIGS. 14A and 14B. The first electric field gradient 21′ applied in step S101 has a relatively shallow slope (i.e., the spatial rate of change of E with respect to distance x is smaller) compared to that applied during the subsequent focusing phase. FIG. 14A shows this electric field gradient 21′ and a steeper first electric field gradient 21″ that may be applied during a later step S111 in the focusing phase (S2) over a comparatively narrower first region R1 (S2). Similarly, the second electric field gradient 22′ applied in step S102 is relatively shallow and wide compared to that (22″) applied in a later step S112. Steps S101 and S102 are therefore configured to collect particles over a wide area with a relatively weak concentration, concentrating the particles in what becomes the first region R1 (S2) for the particle focusing phase. A set of steeper electric field gradients is then applied in the manner already described to achieve strong concentration. The duration of steps S101 and S102 may be longer than that of steps S111 and S112 to provide more time for particles over the wide area to move together.

[0166] 13 shows, particle collection steps S101, S102 can be performed only once or can be repeated multiple times until a sufficient proportion of the injected particles have been collected (step S103). As the method moves to the focusing phase, steps S111 and S112 can also be repeated multiple times in the manner described above. However, it is also possible to move continuously from a phase optimized for particle collection (with relatively wide and shallow gradients 21, 22) toward a phase optimized for particle collection (with relatively narrow and steep gradients 21, 22). Indeed, this gradual change can continue throughout the focusing process, if desired, by, for example, changing the slope and / or lateral extent of the gradients 21, 22 every N repetitions of the sequence, where N is an integer greater than or equal to 1.

[0167] 13 can be performed using only one of steps S101 and S102 (and not the other) before proceeding to step S111. That is, a single application of a relatively wide and shallow electric field gradient (e.g., 21′, 22′) can be performed to achieve some particle collection before proceeding to focusing groups of particles.

[0168] Figures 15, 16, and 17 are plots of another computer simulation of how these principles can be employed and further extended. In this example, a particle collection stage S1 is followed by two particle focusing stages S2 and S3, which use progressively narrower and steeper gradients 21, 22 in each stage. Focusing stage S3 is followed by a selective separation phase S4, which will be described in more detail in connection with later examples.

[0169] In this case, the simulations were performed with a mobility of 0.5×10 -8 (m / s) / V / m) and 0.51×10 -8Two particle types, (m / s) / (v / m), were used, which are similar in size to typical biological particles such as antigen molecules. For clarity, only one trace (i.e., one of the bands) is shown in each plot, as these were found to be approximately the same for each band.

[0170] Figure 15 shows the electric field seen by the band with respect to time, which, as noted above, will not be the same as the applied electric field value because particles take a finite time to travel through the chamber and therefore experience the applied electric field at any particular position. Figure 16 shows the position of the band along the major axis x versus time; note that here the time axis extends further than in Figure 15. Figure 17 shows the electric field seen by the band with respect to distance along the major axis x.

[0171] Table 2 below shows the parameters of the electric field sequence applied in each of steps S1-S3, as well as the parameters of the electric field profile used in separation step S4. It should be noted that, in practice, the first and second electric field gradients applied in this scenario were established as part of a compound electric field gradient using the same approach as described above in connection with FIGS. 4A and 4B. Thus, in the table, the parameters given for the first electric field gradient correspond to the positive section of the compound gradient, and those for the second electric field gradient correspond to the negative section. The parameter "electric field oscillation amplitude" (or "jump") refers to the distance along the x-axis that the compound electric field is moved back and forth (or jumps) to achieve the desired oscillation between the first and second electric field gradients in the first region. [Table 2]

[0172] Thus, during the collection phase S1, the first and second electric field gradients 21, 22 are each applied over a first region of width 18.75 mm, and the sequence is repeated four times with these parameters, for a time t a During this stage S1, the particles are collected across a wide first region and oscillated at a reasonably wide amplitude, as best seen in Figures 16 and 17. a and time t b In step S2, the slopes of the first and second electric field gradients 21, 22 are made larger (relative to those in step S1), which is achieved here by narrowing the first region to a width of 8.75 mm, while maintaining the same maximum and minimum E values ​​as in the previous step. It can be seen that both the period of oscillation and the amplitude of oscillation are significantly reduced relative to step S1. The electric field sequence is repeated 15 times with these parameters, during which the band is significantly narrowed. b Upon completion of step S2, the third focusing stage S3 begins. During this stage S3, the same first and second electric field gradients 21, 22 as in step S2 are applied alternately, but with a faster switching rate (i.e., shorter step duration). This restricts the particles to regions of the gradient with higher electric field magnitude, and the particles experience a stronger focusing effect, as can be seen in Figure 15. The sequence is repeated with these parameters for a further 15 cycles (oscillations).

[0173] At the end of step S3, the desired degree of focusing has been achieved, and in this example the band of particles is then subjected to a selective separation step S4, in which a shallower electric field gradient is applied and the particles are translated along their major axis. In this case, the separation field gradient is translated at a rate of 0.015 cm / s over a distance of about 7.5 cm. The separation process is further described below in connection with subsequent examples.

[0174] 18 and 19 illustrate another embodiment of a focusing method that includes a particle collection step before focusing begins. As shown in the flowchart of FIG. 18, in an initial step S101′, an electrostatic field profile 31 is applied across a second portion P2 of the major axis, which includes a first portion P2, as shown in FIG. 19. The electrostatic field profile includes a zero-crossing point 38 where the magnitude of the electric field is zero. Immediately on one side of point 38, the electric field profile 31 has a positive value, and immediately on the other side of point 38, it has a negative value. The slope of gradient 31 may be smaller than those of gradients 21 and 22 applied in the later particle focusing steps S111 and S112. The particle collection electric field profile 31 has a weak focusing effect on the charged particles in the second portion P2, causing them to move toward the zero-crossing point. This brings the particles into the first portion P1 of the major axis x (and its first region R1), where they undergo the focusing process. An electric field sequence as previously described, including at least steps S111 and S112, repeated as many times as necessary, is then performed in portion P1.

[0175] Some examples of suitable apparatus layouts in which the method as described above may be carried out are now described with reference to Figures 20 and 21. In both examples, the chamber 2 takes the form of an open or closed cavity in the surface of a substrate 19, for example made from a polymer, glass, or ceramic. However, many other implementations are possible, including the use of a vessel such as a tube (e.g., a capillary) or an analysis well. In other cases, the chamber may take the form of a wall-less volume of gel (or other sieving matrix), provided that it is sufficiently self-supporting. The electrodes 4 of the electric field generator are typically spaced apart from one another along the direction of the main axis and arranged around the periphery of the chamber 2. Further details on how the electrode arrangement 4 may be implemented are disclosed in WO 2006 / 070176 and WO 2012 / 153108. In the example of FIG. 20 , chamber 2 (which may or may not be elongated, but preferably has a substantially uniform cross-sectional area along its major axis) is provided with at least one input port 5 a, 5 b (two are shown here) and an extraction port 7, each of which is optional. The input ports are configured to accept, for example, charged particles supported in a solution, which can then be injected into chamber 2 through the port by application of an appropriate electric field between the input port and chamber 2 (one or more electrodes for applying this electric field may be provided but are not shown in the drawing). When the device is used to carry out a reaction between two or more substances (i.e., as a reactor), each substance may be input (in the form of charged particles) into a different input port so that mixing occurs upon injection into chamber 2. Alternatively, a sample of the mixed substances may be prepared and placed into one input port (or directly into chamber 2). One or more extraction ports 7 may also be provided for extracting substances from chamber 2. Again, the extraction port typically comprises electrodes (not shown) across which an electric field can be generated which, in use, draws out the desired charged particles.

[0176] Thus, the focusing method described above can be advantageously used to accelerate a reaction between two or more substances, for example, in an apparatus of the type shown in FIG. 20. The term "reaction" (and related words, such as "react") is used throughout this disclosure to include both chemical reactions, such as binding events, and biological interactions. Charged particles of two or more substances are introduced into the chamber, and an electric field sequence is applied along at least a first portion of the major axis x in the manner described in connection with any of the above embodiments. The group of charged particles becomes narrower (i.e., more concentrated) as a result of the applied electric field sequence, and this focusing of different substances enhances the rate of reaction between them due to the coincidence of different particles at the same location over a period of time and the increased concentration of substances (because the volume of space in which the same amount of material resides is reduced). Because different types of particles in any medium other than a vacuum have different mobilities, the particles also separate into bands of similar particles, and these bands move back and forth, preferably oscillate, along the major axis of chamber 2 when the electric field sequence is repeated multiple times, as described above with reference to FIGS. 8-12. As the process progresses, the bands overlap one another during at least a portion of their movement, and preferably cross one another at least once, and preferably multiple times, further enhancing the reaction rate due to an increased probability of collisions occurring between particles.

[0177] When using the disclosed techniques in connection with accelerating reactions, the primary objective is to focus particle groups as much as possible (to increase concentration) and achieve large physical overlap between the respective bands of particles during cycling. To facilitate these results, first, to achieve strong focusing, it is useful to apply an electric field sequence in which the first and second electric field gradients 21, 22 are applied over a fairly narrow first region R1, and the gradient slope is relatively steep, e.g., varying from 0 to at least ±800 V / cm over a distance of 1 cm or less. The exemplary electric field gradients shown in Figures 4A and 4B are suitable for this. Second, to ensure that the bands remain close together during the process, and thus significantly overlap, the band oscillation amplitude is desirably kept small, which can be achieved by controlling the cycle time. For example, a cycle time of approximately 2 seconds results in an oscillation amplitude of 1-2 mm for the particle mobilities listed in Table 1 above.

[0178] When selecting an appropriate cycle time, a balance must be struck between achieving a small oscillation amplitude (requiring a small cycle time) and ensuring that as many reactants as possible presented to the chamber are captured. The latter can be optionally improved by performing a collection step before focusing begins (as described), but alternatively or additionally, it can be enhanced by a longer cycle time, since this provides more time for widely spread particles to move together during each step. Therefore, it can be advantageous to select a "lenient" duration / cycle time (translating into a wide "bucket") to reach molecules from the edge of the chamber, and then allow them to slide down the gradient to a narrower oscillation without reducing the step duration, or to follow a focusing strategy as described elsewhere to keep the steps narrow, and increase the frequency to find the optimal concentration-response sequence for a particular experiment. There may also be hardware limitations to consider, since pulses must jump from one regime to another for the entire duration. If the frequency is too high, electronic distortions may be imposed on the applied electric field shape. Therefore, there may also be a lower limit on frequency imposed by the hardware.

[0179] The degree of acceleration, and ultimately the yield of reaction products, also depends on the number of times the electric field sequence is repeated. The more cycles performed, the more focusing is achieved, thereby allowing the reaction to proceed further in the same time frame (until one or more of the reactants are fully reacted). Thus, whether sufficient focusing is achieved in step S113 of any of the methods of Figures 5, 13, or 18 above (and Figure 22 below) can be a matter of whether the reaction is complete, or at least has reached a desired stage. For example, the electric field sequence of at least steps S111 and S112 can be repeated until at least a predetermined percentage of the primary reactants has reacted, e.g., 50%, 60%, 70%, 80%, 90%, 99%, or even 100%. The number of cycles required may typically be at least 30, preferably at least 50, or even 100 or more. To establish a correspondence between the number of oscillations and reaction progress, one or more runs of the process may be performed in advance (e.g., as part of a calibration process on the same or a corresponding instrument). For example, a test run may be performed in which N oscillations are performed, after which the resulting contents of the chamber are analyzed, e.g., by performing a separation and using a detector to look for the presence or absence of a particular component, preferably a primary reactant. Multiple runs with different values ​​of N may be performed to establish a minimum value for N at which substantially no primary reactant is detected (meaning 100% of the primary reactant has reacted). The method may then be performed on an actual sample with N (or more) oscillations if 100% of the primary component is desired to react, or with a corresponding fraction of N oscillations if a lesser degree of reaction progress is desired (e.g., 0.5 N if only 50% of the reactants are desired to react).

[0180] The reaction method can end with a focusing process, i.e., when a desired number of repetitions of the electric field sequence have been completed or when a predetermined amount of one of the reactants has been used. One or more of the substances contained in chamber 2 can then be extracted, for example, through extraction port 7. However, if more than one substance is present in the chamber at this stage, it is often difficult to selectively extract only one of them, especially if they are actively focused by the applied electric field and therefore placed closely together. For the same reason, it may not be possible to precisely identify which substances are present in chamber 2 after the reaction has occurred, for example, to confirm the presence or absence of a particular reacted compound.

[0181] Thus, after a reaction is complete or reaches a certain stage, it may be desirable to perform a separation process on the contents of Chamber 2. In the separation process, different particle types are separated into separate bands that are spaced apart from one another and can therefore be easily distinguished and identified. For example, separation can be performed by electrophoresis, in which an electric field is applied to move particles under its influence through a sieving matrix, such as a fluid or gel. This generates hydrodynamic forces that act on each particle in opposite directions. The magnitude of the electric force experienced by the fluid depends on the electric field at the particle's location and the charge on the particle. The hydrodynamic force depends on factors such as the nature of the sieving matrix and its specific size and shape. Under the combined forces, each particle moves to a position where the opposing forces balance each other, i.e., an equilibrium position. This position is different for different particle types, and thus the mixture of particles is separated by type. Many different examples of suitable electrophoresis methods exist. For example, in a simple implementation, separation can be performed by placing the contents of Chamber 2 in a separation channel containing a sieving matrix and applying a uniform electric field. However, better results can be achieved by applying a more complex electric field. For example, the electrophoresis techniques disclosed in WO 2006 / 070176 or WO 2012 / 153108 are particularly advantageous. These require the application of an electric field profile (e.g., a gradient), which is then moved along the separation channel. The various bands find equilibrium positions spaced apart along the gradient and eventually move with the electric field as they translate along the channel, becoming better resolved as they do so. The bands can then be individually detected, identified, and / or extracted as needed.

[0182] Although the contents of Chamber 2 can be removed and transferred (e.g., by pipette) to another device for performing the separation, in a preferred embodiment, the channel is incorporated into the same device used to perform the reaction. A first example of a suitable layout for this is shown in FIG. 21 , where both Chamber 2 (where a focusing process occurs to accelerate the reaction, as described above) and a separate, elongated separation channel 15 are formed as separate cavities in a substrate 19. Chamber 2 is fluidly connected to separation channel 15 by a conduit 9, which may be equipped with appropriate electrodes (not shown) to apply an electric field that draws particles from Chamber 2 into separation channel 15. Because Chamber 2 and separation channel 15 are separate from each other, in this embodiment the major axis x of Chamber 2 and the elongation axis of separation channel 15 can be at any relative angle (including parallel or perpendicular) to each other.

[0183] In use, as illustrated by the flowchart of FIG. 22, charged particles of at least two substances are injected into chamber 2 (e.g., using optional input ports 5a and / or 5b), and then, after one or more optional steps for collecting the particles (represented by step S101″), a focusing method is performed on the particles in chamber 2 by applying an electric field sequence of the type described in any of the above embodiments by electric field generator 4 (steps S111-S113). Some or all of the contents of chamber 2 are transferred through conduit 9 to separation channel 15, where they form an injected “plug” of mixed particles. Typically, this may include particles of at least one reaction product and, in some cases, one or more particles of the original reactants. The separation process is then performed (step S120), for example, by applying an appropriate electric field along the elongation axis x′ of separation channel 15 using additional electrodes of electric field generator 4. For example, the electric field may have an electric field gradient of the type disclosed in WO 2006 / 070176 or WO 2012 / 153108 and may be moved along the elongation axis x' as described herein. Finally, each band of similar particles is separated from the others and spaced apart along the elongation axis x'. The bands may then be detected and identified to confirm the presence or absence of a substance (and thus, for example, a test result). Alternatively or additionally, one or more of the bands may be extracted (step S125) by using the electric field profile to move the selected band to an extraction port (not shown), where the selected band is extracted from the separation channel and the yield of the corresponding material is output.

[0184] The flowchart of Figure 22 is also applicable to other implementations in which a focusing phase occurs before a separation, regardless of whether a reaction is required. When focusing occurs along the same axis as the subsequent separation, this "pre-concentrates" the particles into a narrow volume, so that each band is distinct from the next band at an earlier point in the separation than would otherwise be the case, both in terms of the time it takes and the distance along the separation channel. This significantly reduces the run time of the separation, allowing results to be obtained more quickly, and also allows the overall size of the device containing the separation channel to be reduced.

[0185] An example of a suitable device layout that enables these advantages to be achieved is shown in FIG. 23. Here, the chamber (where focusing occurs) and the separation channel 15 (where separation occurs) are contiguous, each forming part of the same cavity in the substrate 19 and therefore filled with the same sieving matrix. Other components shown in FIG. 23 are the same as those previously described with the same numbers. Again, as previously mentioned, many other physical formats are possible, including providing a tube or other container containing two volumes. Importantly, the elongation axis of the separation channel 15 is aligned with the major axis x of the chamber 2 (i.e., they are parallel and there is no substantial offset). In this example, there is no physical distinction between the chamber 2 and the separation channel 15; in fact, where one ends and the other begins is determined by the nature of the electric fields applied to each, and in fact, their lateral extents can be varied. Note also that the separation process may begin in the chamber 2 before moving into the separation channel 15.

[0186] This is best illustrated in Figures 25A, 25B, and 25C, which are plots showing the electric field applied along the major axis x at different times during the process. Figure 25A shows the situation during the focusing phase. As described above, during this phase, an electric field sequence is applied: a first electric field gradient 21 is applied across a first region R1 in chamber 2 (step S111 in Figure 22), followed by a second electric field gradient 22. As a result, charged particles (including mixtures of multiple types, whether the result of a reaction or corresponding to the mixture originally injected into chamber 2) focus and form an increasingly narrowing band of similar particles. As the sequence is repeated multiple times, the band oscillates within chamber 2. Once a sufficient degree of focusing is achieved, the electric field sequence ends (step S113), and the separation process begins (step S120). As shown in Figures 25B and 25C, this may require the application of an electric field gradient 41 that is translated along the separation channel in the direction of the major axis. Figure 25B shows the applied electric field profile at a first instance t1 during step S120, while Figure 25C shows the applied electric field profile at a later time t2, still during step S120. As explained in more detail in WO 2006 / 070176, each respective band of similar particles moves to a different equilibrium position along the electric field gradient 41 and ultimately moves with the translating electric field. The electric field movement and gradient shape can be controlled to position each band at a desired position along the axis. The bands can be detected, for example, by imaging the channel 15 and / or one or more can be extracted at (optional) port 7.

[0187] Although the separation channel 15 is shown here as straight, this is not required and a curved (e.g., circular) separation channel, arranged as shown in Figure 24, for example, can be used instead. Here, chamber 2 is positioned tangent to the circular separation channel. This "closed loop" channel allows separation to continue as particles circulate through the device. Suitable configurations of separation channels and applied electric fields are disclosed in WO 2006 / 070176.

[0188] The results of computer simulations showing the behavior of particles undergoing the type of separation process described above are described here and contrasted with a comparative example based on the principles provided in WO 2006 / 070176. Figure 26A shows the distance of particles from the equilibrium position with respect to time. Figure 26B shows the distance between bands (i.e., resolution) with respect to time. Figure 27A shows the band width with respect to the distance traveled along the major axis x. Figure 27B shows the band width with respect to time. In each plot, the dashed line shows the results of a simulation according to the comparative example, while the solid line shows the results of a simulation using the method of the present disclosure. In both cases, the layout of chamber 2 and separation channel 15 was the same as shown in Figure 23 above. A mixture of two particle types was injected into chamber 2 at time zero. The mobilities of the particles were 0.5 x 10, respectively. -8 (m / s) / (V / m) and 0.51×10 -8 The mobilities were (m / s) / (V / m), which are typical mobilities of biological particles. Only one trace is shown for each simulation in each figure, because the behavior of each band is nearly identical within each respective simulation in Figures 26A, 27A, and 27B. Figure 26B is a measure of the distance between the two bands (measured as the standard deviation σ of the particle distribution) expressed in terms of a multiple of the width of one of the bands, thus effectively capturing data from both bands.

[0189] In a comparative example, an electric field gradient similar to that shown in Figure 25B was immediately applied, causing translation along the major axis. The simulation used an electric field gradient extending from +800 V / cm to -800 V / cm over 62.5 mm along the x-axis, moving at 0.015 cm / s over a distance of approximately 7.5 cm. As shown in Figure 26A (dashed line), this caused the bands to move toward an equilibrium position, which they reached at approximately t = 50 s, and then remained at equilibrium for the remainder of the separation (during which the equilibrium position moved along the x-axis with the translation of the electric field gradient). However, upon reaching equilibrium, as can be seen from Figure 26B, the first band further merged with the second band, where the distance between the two bands at approximately t = 50 s is significantly smaller than the width σ of each band. Figures 27A and 27B (dashed lines) also show that the band widths are not significantly reduced from their original size at this stage. It is believed that the bands are not resolved from one another until the distance between the bands is at least 2σ (i.e., twice the width of one of the bands), which is approximately 240 seconds after the start of the process, t = t* prev occurs in.

[0190] In a simulation performed according to an embodiment of the disclosed technique, upon injection of two particle types into the chamber, the particles first undergo a focusing process involving the application of the electric field sequence described above, repeated multiple times ( FIG. 25A ). The first electric field gradient 21 extended from +800 V / cm to 0 V / cm over 8.75 mm. The second electric field gradient 22 extended from 0 V / cm to -800 V / cm over 8.75 mm. In practice, this was achieved using a compound gradient that was moved back and forth along the major axis by 5 mm (i.e., the electric field oscillation amplitude was 5 mm). The step duration was 1.25 s. This resulted in a cycle time of 2.5 s. This led to a typical particle oscillation amplitude of approximately 3 mm. The sequence was repeated 30 times. The resulting oscillation of the particles is seen in FIG. 26A (solid line), which continues until approximately t=75 s. During this phase, the particles oscillate back and forth along the major axis, with each band narrowing as described above. This can be seen in Figures 27A and 27B (solid lines).

[0191] The electric field sequence is then stopped, and the separation step is initiated by applying an electric field gradient, which is then translated along the axis (FIGS. 25B, 25C). The parameters during this step were the same as those applied in the comparative example: an electric field gradient extending from +800 V / cm to -800 V / cm over 62.5 mm along the x-axis, moving at 0.015 cm / s over a distance of approximately 7.5 cm. The result can be seen in FIG. 26A (solid line) from approximately t=75 s; that is, the bands move toward their respective equilibrium positions. However, as shown in FIG. 26B, the two bands now separate at t=t*, which is approximately 110 s. new(i.e., spaced apart from each other by at least 2σ). Thus, even at this early time in the process, the two bands are clearly distinct from each other and remain so as they continue to move toward their equilibrium positions. This allows the bands to be distinguished from this point onward, and separation "races" significantly faster at this point than in the comparative example. Overall, testing has shown that using the techniques of the present disclosure, separation can be achieved in less than half the time of conventional methods, and in some cases, in one-third or one-quarter of the conventional duration. This is because the separation phase occurs on the already-focused bands rather than on the original (wide) injection plug. This not only significantly reduces run time, but also allows for significantly shorter chips (especially in the case of linear implementations) because separation requires less distance along the major axis to "complete."

[0192] It should be appreciated that a partial purpose of the initial focusing phase is the opposite of the separation step, i.e., to bring all bands / groups to approximately one point along the major axis x and concentrate them there before proceeding to the second stage of separation. In the two-stage separation approach, the first focusing step provides a head start to the separation step because separation begins with already concentrated bands. Thus, separation occurs significantly more quickly, before the bands reach their respective equilibrium points.

[0193] To illustrate some of the benefits, and now with reference to Figure 32, test data obtained performing embodiments of the invention (and contrasted with the prior art) in the form of assays for detecting Covid-19, Lassa fever, and influenza, respectively, are provided below.

[0194] The simulations already described above with respect to Figures 8-12 also demonstrate the benefit of performing a focusing process (as disclosed herein) prior to separation. Here, after oscillating the band about 2 cm along the major axis x, as described above, a separation gradient is applied beginning at about t = 75 seconds. Figure 8 shows that clear separation is achieved by the time the band reaches about 2.5 cm along the major axis x, at about t = 80 seconds. At this point, the six trajectories are not parallel, meaning they are still in transition and have not yet reached equilibrium (at least not all of them). This allows for a separation runway of about 0.5 cm compared to ~4.5 cm in conventional methods. This provides a separation channel that is nine times shorter than previously required.

[0195] It should be appreciated that the preferred parameters of the electric field sequences described in the examples herein depend on the desired results. When a focusing process is used solely to accelerate a reaction, the primary objective is to achieve maximum concentration of material and to match particles as closely as possible to obtain a high reaction yield. Therefore, to achieve this, the sequence may be repeated many times, e.g., 100 or more oscillations. In contrast, when a focusing process is used to concentrate groups of particles prior to separation, the primary objective is to minimize the focusing time in order to proceed to separation as quickly as possible while still achieving sufficient concentration to also shorten separation time. For example, it has been found that applying 20-40 (e.g., 30) oscillation periods achieves a good reduction in overall run time (and separation distance required). In cases where a focusing process is used to accelerate a reaction and as a pre-concentration step for separation (in which reaction products are separated), a balance between the two factors is required, and some intermediate oscillation frequency (e.g., 50-80) may prove optimal.

[0196] In alternative embodiments, similar principles can be applied to achieve a lesser degree of band focusing before initiating separation. These still result in shorter run times compared to conventional techniques. For example, rather than applying both the first and second electric field gradients 21, 22 (sequentially) in the manner described above, some degree of focusing can be achieved if only one such gradient is applied before initiating separation. That is, either the first electric field gradient 21 or the second electric field gradient 22 (but not both) can be applied once, immediately followed by separation. In this case, band oscillation does not occur. The electric field gradients 21, 22 used for focusing are preferably relatively steep relative to those used during the subsequent separation step (41). The change from one gradient to the other can be discrete or continuous. For example, if the first electric field gradient 21 is used for focusing, it can then begin to move along the channel, thereby initiating separation, while gradually decreasing the slope and optimizing separation as the gradient moves along the remainder of the channel. The gradual change in slope can, in some cases, continue throughout the separation.

[0197] Another alternative example is shown in Figures 28A, 28B, and 28C. In this case, initial particle focusing is performed by applying an electrostatic field 31 having a zero-crossing point, as shown in Figure 28A. This is similar to the particle collection step S101' described above. The particles move toward the zero-crossing point located in a first region of chamber 2, thereby concentrating the group of particles to some extent. The separation step S120 can then be initiated (Figures 28B, 28C), for example, by applying a gradient 41 that is translated along the separation channel as described above. In this example, no oscillation of the bands occurs. The transition between the focusing phase S101' and the separation phase S120 can be discrete or continuous, as in the previous example. Again, due to the increased focusing of the bands at the beginning of the separation, the separation is completed more quickly than in conventional techniques.

[0198] Thus far, the examples presented have been described in the context of a scenario in which all particles of interest have the same charge sign. That is, all particles are either positively or negatively charged (however, their charge magnitudes are usually different unless all particles in the sample are of the same type). This is often preferable, especially when focusing is used to promote reactions between particles of different materials (because, as will be explained, this allows the bands to be more intimately mixed). Generally, it is possible to configure all particles to have the same charge sign (in fluid-based implementations), for example, by controlling the pH of the solution in which the particles are supported. However, the techniques according to the present invention can be applied to other situations in which the initially injected particles are of mixed charge signs (i.e., some positive, some negative).

[0199] For context, FIG. 29 illustrates the case where, in the first step S111 of the electric field sequence, a mixed set of particles 10a, 10b, 10c, and 10d are subjected to a first electric field gradient 21 of the type described in connection with the first embodiment (FIG. 3A) above. The negative particles 10a, 10b move in the +x direction, focus, and form a first group 12a in the manner previously described. However, any positive particles in the same first region R1, such as particles 10c and 10d shown here, move in the opposite (−x) direction and thus do not form part of group 12a. Furthermore, as the particles move in the −x direction (as indicated by the arrows in FIG. 29), they experience an increasing electric field magnitude, resulting in particles 10c and 10d becoming further dispersed from one another during the step. Typically, particles are accelerated out of region R1 and do not form groups. This may not be a problem if the particles in question are not of interest.

[0200] However, in other cases, it may be desirable to concentrate both positive and negative particles. Examples of how this is possible are described with reference to Figures 30 and 31. Essentially, these involve simultaneously implementing electric field sequences of the type already described in two laterally offset regions along a major axis, with the direction of the electric field gradient in each region reversed relative to one another. In this way, one region can be used to concentrate positive particles, while simultaneously the other concentrates negative particles.

[0201] FIG. 30A shows an exemplary electric field profile that may be applied along axis x in the first step (S111) of such a sequence. In a first region R1, a first electric field gradient 21 is applied as already described above. Simultaneously, in a second region R2, a third electric field gradient 23 is applied. All points in the third electric field gradient 23 have the same sign (e.g., positive in this example) as in the first electric field gradient 21, but the electric field magnitude decreases in the opposite direction along the major axis x compared to the first electric field gradient 21. That is, the direction of the slope is opposite. The first and third electric field gradients 21, 23 together may form a composite electric field profile 29′. This has the result that particles of a first electric field sign (e.g., negative) are focused to form a first group 12a in the first region R1, while particles of the other electric field sign (e.g., positive) are focused to form a second group in the second region R2. In the next step of sequence S112, a second electric field gradient 22 (previously described) is applied in the first region R1, and a fourth electric field gradient 24 is applied in the second region R2. Again, this has the same sign as the second electric field gradient but slopes in the opposite direction, continuing the focusing of the second group 12b. The second and fourth electric field gradients 22, 24 together form another composite electric field profile 29''.

[0202] Steps S111 and S112 may be performed only once, or more preferably, repeated multiple times to achieve a greater degree of focusing. In this case, positive particles oscillate primarily in one region, while negative particles oscillate primarily in another region. For this reason, particles of mutually reactive substances are preferably configured to have the same charge sign. However, it is also possible to configure each group of particles so that the oscillation width increases as it extends into the other's region. If the particles move sufficiently fast through the changing electric field, the groups may not be significantly affected by crossing into the other's region, so that a reaction between the two groups can still occur. Once sufficient focusing is achieved, the contents of the chamber can be extracted and / or the contents of the chamber undergo a separation process in any of the forms previously described.

[0203] An enhancement to the embodiment of FIG. 30 is shown in FIG. 31. During application of the electric field shown in FIG. 30A, it is likely that some of the particles will reach the boundary between regions R1 and R2. When this occurs, some of the negative particles of group 12a will enter second region R2, and if they remain there for a sufficient period of time, they will be accelerated in the +x direction and lost from the focusing process under the influence of third electric field gradient 23. The same can happen with other positive particles that enter first region R1 from second region R2 (but in the −x direction). To reduce the likelihood of this happening, first and second regions R1, R2 are preferably spaced from each other along major axis x by a non-zero distance, forming third region R3. An example of this is shown in FIGS. 31A and 31B.

[0204] It is not necessary to apply an electric field in the third region R3. The space where the electric field magnitude is zero acts as a buffer zone, reducing to some extent the possibility that particles will reach the other region. However, if the electric field is zero in the third region, there is a risk that particles will stop there and not rejoin their respective groups. To avoid this, it is desirable to apply a non-zero electric field in the third region with the same sign as the electric field gradient on each side. Thus, as shown in FIG. 31A, during step S111, a positive non-zero electric field 25 is applied in the third region, and during step S112, a negative non-zero electric field 26 is applied there. Both electric fields in this example are uniform (i.e., have a substantially flat profile) across the third region R3. This is desirable because there is no focusing or dispersion effect on particles of each charge sign here. However, this is not necessary because an electric field profile with a shallow slope has almost no focusing / dispersion effect and can therefore also be used. However, any profile in the third region should be gentler than that adopted in the first and second regions.

[0205] The concentrated bands of positive and negative particles formed in the first and second regions R1, R2 can reach the third region R3 at different times in the sequence, where they can interact with each other. This allows reactions between particles of opposite charge signs to occur through such an arrangement, although the reaction efficiency will likely be lower than that achieved between particles of the same charge sign.

[0206] As previously mentioned, aspects of the present invention provide methods in which at least two substances are brought into proximity with one another using the focusing techniques described herein and, optionally, subsequently undergo a separation step to separate reaction products and / or identify remaining reactants. As noted above, such methods have broad applicability in biomedical fields where it may be important to improve the rate and / or efficiency of colocation, binding interaction, or reaction between two substances.

[0207] Thus, the present invention can be used in any biological assay, protocol, or application where improved reaction rates or improved colocation or specific binding of multiple molecules is desired. For example, the present invention can be used to determine protein-protein interactions, protein-DNA interactions, in pull-down assays, immunoprecipitation assays, high-throughput screening assays, synthetic biology, and / or drug production.

[0208] Understanding how proteins interact with each other and with nucleic acids (e.g., DNA and / or RNA) or other biological molecules (such as polysaccharides and / or lipids) is paramount in the drug development process. Therapeutic drugs are often designed to target specific disease pathways by targeting specific key proteins or genes in those pathways. For example, therapeutic drugs may act to enhance the action of the target protein (agonists) or, alternatively, to interfere with the action of the target protein (antagonists). Therefore, many preclinical studies involve in vitro studies aimed at assessing the interaction of test therapeutic agents with target proteins. In addition, preclinical studies can also be used to identify binding partners of proteins of interest or to generate structural details of protein complexes (Zhou et al., ChemMedChem, 11:738-756, 2016).

[0209] These protein-protein and protein-DNA interactions can be identified and visualized by various methods, such as pull-down analysis, immunoprecipitation analysis, fluorescence polarization, surface plasmon resonance, nuclear magnetic resonance, circular dichroism analysis, static and dynamic light scattering, analytical ultracentrifugation, isothermal titration calorimetry, or microscale thermophoresis. However, these methods have many drawbacks, including long analysis times, high sample consumption, and low throughput (Zhou et al., ChemMedChem, 11:738-756, 2016).

[0210] The present invention provides improved methods that overcome the aforementioned drawbacks. For example, in determining protein-protein interactions, the methods disclosed herein can be used to focus a protein of interest with a test agent to assess the level of binding. Alternatively, the methods disclosed herein can be used to detect the presence of one protein through the use of another protein. That is, the methods disclosed herein can be used in analyte detection. As a result of the disclosed techniques, rapid focusing between the protein of interest and the test agent / binding protein is achieved. This increases the concentration of each of the respective substances at a specific point. This increased collocation can serve to accelerate any reaction between or involving the two. Alternatively, rapid collocation can enhance binding, for example, where analyte detection is required. Optionally, the methods disclosed herein can further include an electrophoresis step that can separate and / or visualize the final product, e.g., a protein-drug complex or a protein-protein complex, from the individual starting materials. Additionally, a selective electrophoresis step may be used to identify unbound proteins (i.e., those not present in the complex), which may then be used as a measure or indication of the level of binding that has occurred between the protein of interest and the test agent / binding protein.

[0211] An additional method that can identify and / or detect physical interactions between proteins is an in vitro technique known as "pull-down" analysis. Typically, this method involves the use of affinity purification with various washing and elution steps, in which a "bait" protein is used to separate other proteins in a sample. Following the "pull-down" step, protein fractions are separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then visualized using common methods such as gel staining and Western blotting. However, performing the aforementioned electrophoresis steps after a "pull-down" experiment can be very time-consuming and laborious for users. This was demonstrated in Louche et al., 2017 (Methods in Molecular Biology, 1615:247-255), who provided a "pull-down" protocol. This protocol details a method with over 2.5 hours of pre- and post-incubation steps and a 2.5-hour incubation step, totaling over 5 hours of experimental steps (not including the subsequent electrophoresis step).

[0212] The invention disclosed herein overcomes these drawbacks of the pull-down method. The techniques disclosed herein eliminate the need for nearly all sample preparation steps and further reduce incubation times to a fraction of the time outlined above (e.g., about 1 minute or less). Therefore, the methods disclosed herein can be used to significantly shorten the time required to reach an analysis of protein-protein interactions and reduce the number of reagents required to successfully complete an assay. Sample preparation is expected to involve simply contacting the sample with an antibody solution, which can then be used directly in the device of the present invention; i.e., introduced to the chip's receptor and then electrokinetically injected into the channel. Prolonged pre-incubation of the antibody with the sample is expected to be unnecessary. It is also expected that all steps can be performed at room temperature. Therefore, the methods of the present invention omit multiple steps involved in typical immunoassays, such as ELISA, surface antigen immobilization, blocking, washing, primary antibody incubation, washing, secondary antibody incubation, washing, subsequent visualization, or alternatively, column-based precipitation steps. A further advantage of the present invention is that the binding of antibodies to analytes occurs in solution rather than on a surface, resulting in a more natural setup and more accurate results, especially in low affinity protein binding assays.

[0213] Thus, the present invention also provides methods disclosed herein as alternatives to "pull-down" assays. Thus, the methods disclosed herein can be used to confirm the presence of protein-protein interactions in a particular sample predicted by other techniques, or as initial screening assays to identify previously unknown protein-protein interactions. Furthermore, the methods disclosed herein can be used to detect the activation state of specific proteins. For example, proteins that are activated in response to tyrosine phosphorylation can be co-incubated with SH2 domains. These domains target phosphotyrosines on specific proteins. Thus, the methods disclosed herein can be used to promote interactions with phosphorylated proteins and thereby identify phosphorylated proteins. The methods disclosed herein may further include an electrophoresis step that can distinguish and separate reaction products.

[0214] Another commonly used technique in the biomedical field is immunoprecipitation. This technique is similar to the "pull-down" method mentioned above, except that immunoprecipitation is based solely on antibody-antigen interactions. This technique uses antibodies that specifically bind to a particular protein to precipitate protein antigens from solution. Immunoprecipitation methods come in various formats, including individual protein immunoprecipitation, protein complex immunoprecipitation, chromatin immunoprecipitation, and RNP immunoprecipitation. Various methods can be applied after an immunoprecipitation assay to further analyze the interaction of interest. For example, Perez et al., 2007 (Methods in Molecular Medicine, 131:123-139), described the use of chromatin immunoprecipitation followed by an electrophoretic mobility shift assay to study DNA-binding protein interactions in vitro. However, similar to the drawbacks of the "pull-down" assay mentioned above, the disclosed protocol requires many tedious steps and significant incubation times. In contrast, the present invention provides a time-efficient and significantly streamlined protocol, allowing users to achieve desired results in a shorter time. Thus, the present invention also discloses the methods disclosed herein as alternatives to immunoprecipitation assays.

[0215] The drug development process takes years and involves many stages. One critical step in this process is conducting high-throughput screening of drug candidates to narrow down the most promising candidates for further development and research. As previously discussed, the inventions disclosed herein significantly reduce the time required to assess the level of coexistence or specific binding of target molecules and significantly reduce the amount of reagents required compared to the typical biological assays described above, features particularly advantageous in the context of high-throughput screening. Accordingly, the present invention also discloses methods disclosed herein for use in high-throughput screening assays. That is, the methods of the present invention use the focusing technology described herein to bring at least two substances into close proximity with each other and can be used to rapidly assess the ability of a drug candidate to bind to a drug target molecule (e.g., a protein) or to assess the activation / inactivation of biological activity associated with the drug target molecule. Furthermore, the methods disclosed herein increase the speed at which two substances come into contact with each other, allowing for a more time-efficient process. If necessary, the methods may further include an electrophoresis step to identify / isolate reaction products.

[0216] Those skilled in the art will readily appreciate that all of the above biologically relevant assays play an important role in the drug discovery process. However, the methods described herein significantly improve the ability to perform biological assays more rapidly, resulting in a more streamlined drug discovery process and more efficient identification of successful drug candidates.

[0217] Exemplary test data obtained from performing assays for Covid-19, Lassa fever, and influenza using methods according to embodiments of the present invention are described with reference to Figure 32 et seq. and compared with conventional methods.

[0218] Figures 32A and 32B are graphs showing the results of two Covid-19 tests performed using the method of the present invention. In the first run (Figure 32A), the sample was negative (i.e., this was a control run), while in the second run (Figure 32B), the sample was positive and "spiked" with a known concentration of SARS-CoV-2 antigen. In both cases, the samples were prepared as follows: Covid-19-negative swab samples from machine users were collected according to standard lateral flow device (LFD) protocols and diluted in 100 μl of viral transport medium (Neuromics VTM). The swab samples were then mixed with a rabbit anti-SARS-CoV-2 nucleocapsid protein polyclonal antibody at a concentration of 100 ng / μl and 20% Ficoll 400. The antibody had previously been tagged with Alexa Fluor 488. For the "spiked" sample (Figure 32B), purified nucleocapsid antigen [recombinant SARS-CoV-2 nucleocapsid protein, RayBiotech] was added to the mixture to achieve a 1:1 billion dilution of the stock antigen sample. This corresponds to 100 molecules in the final solution. After mixing with a pipette, the entire mixture was transferred to the injection well of the device as described above and diluted one last time with Tris-glycine buffer (pH 8.23 ​​at 25 °C) before injection. No denaturants were added, allowing the protein to run in its native folded form. The sample was injected into the separation channel by electrophoretic injection.

[0219] Next, an electric field sequence was applied according to the techniques described above to focus and mix the particles within the sample, vibrate the particles, and promote binding reactions between the antibody and the target antigen present in the sample. The electric field parameters are shown in Table 3 below and include a static collection phase S1 (no vibration) followed by a focusing phase S2, during which vibration occurs, involving a change in the electric field gradient at each step of the sequence. The first and second electric field gradients were provided by a combined electric field gradient (as in the example in Figure 4) and were controlled to jump back and forth along the major axis in the manner described above. At the end of the vibration phase, the chamber contains unreacted antibody ("Ab") and / or bound complexes ("Ab+Ag"). Complexes, of course, only form if the target antigen SARS-CoV-2 is present in the sample. A certain amount of unreacted antibody ("Ab") will remain after the reaction, regardless of whether the target antigen SARS-CoV-2 was found in the sample, because the antibody is usually added in a sufficient amount so that some remains unbound. A separation step S3 is then performed, separating the material into bands that migrate along the separation channel as described above.

[0220] Detectors, such as fluorescence detectors, are positioned at predetermined locations along the separation channel. As each band passes through the detector, the detector outputs a signal, for example, based on the fluorescence detected if each band carries a fluorophore. (Alternative detection methods, such as impedance detection or absorption imaging, can be used.) As the bands are separated along the channel, each band reaches the detector at a different time. Figures 32A and 32B show the signal output by the detector versus time in terms of the number of scans (each scan corresponds to 100 ms). Leading bands produce signals earlier, and trailing bands produce signals later. Usually (but not always), smaller molecules (e.g., antibodies, Ab, in an immunoassay) lead, and larger molecules (e.g., Ab+Ag complexes) follow. In Figure 32A, one peak is seen at approximately 6250 scans (approximately t = 10 min), which is the Ab-only peak (because this was the negative control run). In Figure 32B, in addition to the Ab-only peak, a second peak is seen, marked with an arrow "C" at approximately 7750 scans (approximately t = 13 minutes), which is the complex (Ab + Ag) peak. The presence of the second peak C, corresponding to the complex, indicates a positive Covid-19 test. It is noteworthy that the technique successfully detected just 100 molecules of SARS-CoV-2 nucleocapsid antigen, a significantly lower concentration than can be detected using conventional methods. This demonstrates the enhanced sensitivity of the disclosed technique. [Table 3]

[0221] It can be seen from Figures 32A and 32B that the run took approximately 13 minutes in total to provide a positive result (measured from the beginning of step S1). This could be shortened by moving the detector closer to the injection point in the device, allowing the band to reach the injection point sooner. However, this duration is still many times faster than conventional PCR testing techniques, which require approximately 100 minutes (35 x 3-minute cycles) to achieve the highest sensitivity. Conventional PCR testing techniques have not yet been able to detect such low concentrations of antigen as demonstrated here. Furthermore, because the disclosed technology has such increased sensitivity, little or no sample incubation time (1 minute or less) is required prior to injection of the sample into the device. This reduces the overall time required to run the assay very substantially.

[0222] In another experiment, the Covid-19 assay was run without the shaking step. The results are shown in Figure 33. In this run, the (spiked) sample was prepared in the same format as described immediately above, but prior to injection, the sample was incubated at room temperature for 4 hours in a 3:1 excess antibody stock concentration. This resulted in the injected sample containing approximately 1000 antigen molecules (i.e., 10-fold that provided in the test in Figure 32B, which is still an extremely high dilution). This was necessary to attempt to obtain a positive detection result due to the lower sensitivity of the assay technology used in Figure 33.

[0223] Upon injection of the sample into the chamber, the device was controlled to perform a collection stage S1, followed by a static concentration stage S2 (without vibration) and then a separation stage S3. The applied parameters for each stage are shown in Table 4 below. [Table 4]

[0224] It can be seen from Figure 33 that due to the broader band of particles (less concentration achieved), the detection signal contains significantly more noise than in the case of Figures 32A and 32B. The antibody peak is still clearly visible at approximately 7000 scans, but the complex peak (labeled C) is only just noticeable, despite the sample containing a significantly higher antigen concentration than that used in the test of Figure 32.

[0225] Next, a method according to an embodiment of the invention was applied to a Lassa fever immunoassay using the purified antigen. The results are shown in Figures 34A and 34B. To prepare the sample, 100 μl of mouse anti-Lassa fever virus GP1 polyclonal antibody (concentration 1000 ng / μl) was first mixed with 10 μl of the modification reagent from the Abcam Alexa Fluor 488 Conjugation Kit to label the antibody. The entire mixture was then transferred to the lyophilized material, resuspended, and incubated for 15 minutes in the dark. After the incubation, 10 μl of quencher reagent was added, mixed, and incubated for an additional 5 minutes. The sample was then aliquoted before storage. Next, the labeled antibody sample was diluted 1:10 (100 ng / μl) and mixed with 7 μl of distilled H2O and 2 μl of 20% Ficoll 400. For a control run, this mixture was spun for 1 minute and transferred to the injection well of the chip. The samples were finally diluted once more with 100 μl of Tris-glycine buffer (pH 8.23 ​​at 25° C.) and analyzed.

[0226] To provide a "spiked" (positive) sample, 1 μl of recombinant Lassa virus GP1, C-terminal mouse Fc tag (concentration 1000 ng / μl) was diluted with 19 μl of distilled H2O to achieve a dilution factor of 1:20 (50 ng / μl). 1 μl of the diluted sample was then mixed with 1 μl of diluted antibody and 1 μl of 20% Ficoll 400. This mixture was spun for 1 minute and transferred to the injection well of the chip. The sample was finally diluted once more with 100 μl of Tris-glycine buffer (pH 8.23 ​​at 25°C) and analyzed.

[0227] The analysis performed on both the control and spiked samples was a three-step process, including a collection step S1, a vibration focusing / reaction step S2, and a separation step S3. The applied parameters were the same as those shown in Table 3 above. In the control run, as shown in Figure 34A, a single peak corresponding to the antibody was obtained (i.e., a negative result). As shown in Figure 34B, the antibody-only peak (at approximately 6400 scans) and the complex peak C (at approximately 7000 scans) are clearly distinguishable, indicating a positive test result. The intensity of the complex peak C is much more pronounced than when using conventional techniques. Conventional techniques typically require the sample to be kept at room temperature at stock concentrations for at least one hour, preferably overnight, to comprehensively incubate such samples.

[0228] Finally, the effectiveness of the technique was also demonstrated in influenza B analysis. The results are shown in Figure 35. To prepare the (spiked) sample in a test tube, 6 μL of dH2O, 2 μL of 25% Ficoll 400, 1 μL of influenza B antigen (IVB Ag) [70 ng / μL], and 1 μL of labeled antibody (Lab) [100 ng / μL] were mixed before 2 μL of the solution was pipetted into the injection well of the device. The sample was then diluted in approximately 100 μL of Tris-glycine buffer and analyzed. The analysis performed was a three-step process, including collection step S1, vibration focusing / reaction step S2, and separation step S3. The applied parameters were the same as those listed in Table 3 above. As shown in Figure 35, the antibody-only peak (at approximately 6400 scans) and complex peak C (at approximately 6750 scans) could be clearly distinguished, indicating a positive test result. Note that here, complex peak C has a significantly greater intensity than the antibody-only peak. This is due to a larger proportion of the antibody probe reacting with the antigen compared to the situation in the Covid-19 and Lassa fever assays. This may be due to the relatively smaller amount of antibody used in, for example, the influenza assay.

Claims

1. 1. A method for focusing charged particles, comprising: a) injecting charged particles of at least one substance into a chamber having a major axis; b) applying an electric field sequence along at least a first portion of the major axis of the chamber, wherein applying the electric field sequence comprises: b1) applying a first electric field gradient, the sign of the electric field being either positive or negative substantially everywhere within a first region of the major axis, and the magnitude of the electric field decreasing in the first region along the major axis in the first direction of the major axis; b2) applying a second electric field gradient, the sign of the electric field being either positive or negative substantially everywhere within the first region of the major axis, and the magnitude of the electric field decreasing in the first region along the major axis in a second direction of the major axis opposite the first direction; during step (b1), the group of charged particles moves along the major axis in the first direction under the influence of the first electric field gradient, and in step (b2), the group of charged particles moves along the major axis of the chamber in the second opposite direction under the influence of the second electric field gradient, and during each of steps (b1) and (b2), a leading edge of the group moves slower than a trailing edge of the group due to the smaller electric field magnitude acting on the particles at the leading edge, thereby gradually reducing the width of the group of charged particles.

2. 2. The method of claim 1, wherein the electric field sequence switches between steps (b1) and (b2) without an intermediate transition step, preferably with substantially no time lapse between steps (b1) and (b2).

3. 2. The method of claim 1, wherein the electric field sequence further comprises a transition step (b1') between step (b1) and step (b2), during which the first electric field gradient is changed to the second electric field gradient, and wherein the duration of the transition step (b1') is shorter, preferably at least 10 times shorter, more preferably at least 50 times shorter, and most preferably at least 100 times shorter than the duration of each of step (b1) or step (b2).

4. 4. The method of claim 1 or 3, wherein the electric field sequence further comprises a transition step (b2') after step (b2), during which the second electric field gradient is changed to the first electric field gradient, and wherein the duration of transition step (b2') is shorter, preferably at least 10 times shorter, more preferably at least 50 times shorter, and most preferably at least 100 times shorter than the duration of each of steps (b1) or (b2).

5. 5. The method of claim 1, wherein during at least a portion of step (b1), the first electric field gradient is translated in the first direction along the major axis of the chamber within the first region, and / or during at least a portion of step (b2), the second electric field gradient is translated in the second direction along the major axis of the chamber within the first region.

6. 6. The method of claim 1, wherein the electric field sequence is repeated multiple times, preferably at least 3 times, more preferably at least 10 times, even more preferably at least 30 times, and most preferably at least 50 times.

7. In step (b), the first and second electric field gradients each define a compound electric field profile along the first portion of the major axis of the chamber, the compound electric field profile comprising: a first portion corresponding to the first electric field gradient, the sign of the electric field being either positive or negative at substantially all points and the magnitude of the electric field decreasing in the first direction of the major axis; a second portion corresponding to the second electric field gradient, the sign of the electric field being either positive or negative at substantially all points and the magnitude of the electric field decreasing in the second direction of the major axis opposite the first direction; 7. The method of claim 1, wherein applying the electric field sequence comprises varying a position of the first portion relative to a first axis by applying the compound electric field profile at different positions along the major axis of the chamber in steps (b1) and (b2), respectively, such that during step (b1), the first portion of the compound electric field profile is applied along the first region of the major axis of the chamber, and during step (b2), the second portion of the compound electric field profile is applied along the first region of the major axis of the chamber.

8. 8. The method of claim 7, wherein the electric field sequence further comprises a transition step (b1′) between steps (b1) and (b2), during which the first electric field gradient is changed to the second electric field gradient in the first region, the duration of the transition step (b1′) being shorter than the duration of each of steps (b1) or (b2), and during which the first electric field gradient is changed to the second electric field gradient in the first region by moving the compound electric field profile along the major axis.

9. 9. The method of claim 7 or 8, wherein the electric field sequence further comprises a transition step (b2') after step (b2), during which the second electric field gradient is changed to the first electric field gradient in the first region, the duration of the transition step (b2') being shorter than the duration of each of steps (b1) or (b2), and wherein during the transition step (b2'), the second electric field gradient is changed to the first electric field gradient in the first region by moving the compound electric field profile along the major axis.

10. 10. The method of claim 8 or 9, wherein the electric field sequence is repeated multiple times by successively moving the compound electric field profile in alternating directions along the major axis.

11. 11. The method of claim 1, wherein all of the charged particles injected in step (a) are positively charged or all of the charged particles injected in step (a) are negatively charged.

12. In step (b1), simultaneously with applying the first electric field gradient in the first region, a third electric field gradient is applied in a second region of the major axis laterally displaced from the first region, wherein the sign of the electric field is the same as the sign of the first electric field gradient substantially everywhere in the second region, and the magnitude of the electric field increases in the second region along the major axis in the first direction; In step (b2), simultaneously with applying the second electric field gradient in the first region, a fourth electric field gradient is applied in the second region, wherein the sign of the electric field is the same as the sign of the second electric field gradient substantially everywhere in the second region, and the magnitude of the electric field increases in the second direction along the major axis in the second region; 7. The method according to claim 1, wherein during step (b1), in the first region, a first group of the charged particles having a first charge sign moves in the first direction along the major axis under the influence of the first electric field gradient, while in the second region, a second group of the charged particles having an opposite charge sign moves in the second opposite direction along the major axis under the influence of the third electric field gradient; and during step (b2), in the first region, the first group of the charged particles moves in the second direction along the major axis under the influence of the second electric field gradient, while in the second region, the second group of the charged particles moves in the first direction along the major axis under the influence of the fourth electric field gradient, and during each of steps (b1) and (b2), a leading edge of each group moves slower than a trailing edge of the group due to the smaller electric field magnitude acting on the particles at the leading edge, thereby continuously reducing the width of each group of charged particles.

13. The first and second regions of the major axis are spaced from one another by a third region of non-zero width, preferably in step (b1), simultaneously with applying the first and third electric field gradients, a non-zero electric field of the same sign as the signs of the first and third electric field gradients is applied in the third region, the non-zero electric field preferably having a spatial rate of change of electric field as a function of distance along the major axis that is less than that of the first and third electric field gradients, most preferably substantially constant across the third region; 13. The method of claim 12, wherein in step (b2), simultaneously with applying the second and fourth electric field gradients, a non-zero electric field of the same sign as that of the second and fourth electric field gradients is applied in a third region, the non-zero electric field preferably having a spatial rate of change of electric field as a function of distance along the major axis that is less than that of the second and fourth electric field gradients, and most preferably is substantially constant across the third region.

14. 14. The method of claim 1, wherein the first and second electric field gradients have substantially the same spatial rate of change of the electric field as a function of distance from one another along the major axis.

15. 15. The method of claim 1, wherein the first and second electric field gradients have substantially the same maximum and minimum electric field magnitudes.

16. 16. The method of claim 1, wherein the first and second electric field gradients have substantially the same profile shape as each other.

17. 17. The method of claim 1, wherein the first and second electric field gradients are monotonic along at least the first region of the major axis.

18. 18. The method of claim 1, wherein the first and second electric field gradients are substantially linear or curved, such as parabolic or exponential.

19. 19. The method of claim 1, wherein steps (b1) and (b2) have substantially the same duration as each other.

20. 20. The method of any one of claims 1 to 19, wherein the electric field sequence is repeated multiple times, and wherein steps (b1) and (b2) together occupy at least 80%, preferably at least 90%, more preferably at least 99% of the duration of each cycle.

21. 21. The method according to any one of claims 1 to 20, wherein the electric field sequence consists of steps (b1) and (b2) plus optional transition steps (b1') and / or (b2'), and the electric field sequence is preferably repeated multiple times.

22. 22. The method of any one of claims 1 to 21, wherein the electric field sequence has a total duration of 0.1 to 100 seconds, preferably 0.1 to 10 seconds, more preferably 0.1 to 5 seconds, and most preferably 1 to 3 seconds.

23. 23. The method of any one of claims 1 to 22, wherein in step (b), the electric field sequence is repeated a plurality of times, the first and second electric field gradients and the duration of each cycle being configured to cause the charged particles to oscillate along the major axis of the chamber, the amplitude of the oscillation being in the range of 0.1 to 10 mm, preferably 0.5 to 5 mm, more preferably 1 to 3 mm.

24. 24. The method of any one of claims 1 to 23, wherein the first region of the main axis of the chamber has a length in the range of 0.1 to 5 cm, preferably 0.5 to 2.5 cm, more preferably 0.5 to 1.5 cm.

25. 25. The method of claim 1, wherein in step (a), the charged particles are of a plurality of materials and comprise charged particles of different mobilities, and during step (b), the groups of charged particles are additionally formed into a plurality of bands under the influence of the electric field sequence, each band comprising charged particles with the same mobility, and the width of each band being successively reduced.

26. 26. The method of any one of claims 1 to 25, wherein in step (b), the electric field sequence is repeated a plurality of times and the size of the first and / or second regions is reduced as the method progresses by reducing the width of the major axis along which each electric field gradient is applied at least once between successive cycles, preferably every N cycles, where N is an integer greater than or equal to 1.

27. 27. The method of claim 26, wherein step (b) may include a particle collection phase followed by a particle focusing phase, and wherein the size of the first and / or second regions is larger in the particle collection phase than in the particle focusing phase.

28. 28. The method of any one of claims 1 to 27, wherein the spatial rate of change of electric field magnitude as a function of distance along the major axis of each electric field gradient is increased as the method progresses, and is preferably lower in a particle collection stage than in the subsequent particle focusing phase.

29. After step (a) and before step (b), 29. The method of any one of claims 1 to 28, further comprising: (a') collecting the charged particles to position a majority, preferably substantially all, of the group of charged particles within the first portion of the major axis of the chamber.

30. The step (a') a'1) applying an electrostatic field gradient along a second portion of the major axis that is greater than and includes the first portion, the electrostatic field gradient including a zero crossing point origin located in the first portion, the charged particles moving toward the zero crossing point under the influence of the electrostatic field gradient.

31. 1. An apparatus for focusing charged particles, comprising: a chamber into which, in use, charged particles of at least one substance are injected, said chamber having a major axis; an electric field generator configured to apply an electric field along at least a first portion of the major axis of the chamber; a controller configured to control the electric field generator and programmed to control the electric field generator to apply an electric field sequence along at least the first portion of the major axis of the chamber, the electric field sequence comprising: b1) applying a first electric field gradient, the sign of the electric field being either positive or negative substantially everywhere within a first region of the major axis, and the magnitude of the electric field decreasing in the first region along the major axis in the first direction of the major axis; b2) applying a second electric field gradient, the sign of the electric field being either positive or negative substantially everywhere within the first region of the major axis, and the magnitude of the electric field decreasing in the first region along the major axis in a second direction of the major axis opposite the first direction; and wherein, in use, during step (b1), the group of charged particles moves along the major axis in the first direction under the influence of the first electric field gradient, and in step (b2), the group of charged particles moves along the major axis of the chamber in the second opposite direction under the influence of the second electric field gradient, and during each of steps (b1) and (b2), a leading edge of the group moves slower than a trailing edge of the group due to the smaller electric field magnitude acting on the particles at the leading edge, thereby gradually reducing the width of the group of charged particles.

32. 32. The apparatus of claim 31 , wherein the controller is further programmed to control the electric field generator to perform the method of any one of claims 1 to 30.

33. 33. The apparatus of claim 31 or 32, wherein the electric field generator comprises an array of electrodes disposed along at least the first portion of the major axis of the chamber.

34. 34. The apparatus of any one of claims 31 to 33, wherein the chamber is provided with at least one input port for injecting a sample into the chamber, the sample comprising at least the charged particles of at least one substance.

35. 35. Apparatus according to any one of claims 31 to 34, wherein the chamber is provided with at least one outlet port for extraction of groups or bands of charged particles from the chamber.

36. 36. Apparatus according to any one of claims 31 to 35, further comprising a detector adapted to detect groups or bands of charged particles in the chamber, said detector preferably adapted to image said groups or bands.

37. 1. A method for inducing a reaction between at least two substances, comprising: (i) providing charged particles of said at least two materials; (ii) focusing the charged particles in a chamber using the method of any one of claims 1 to 30, wherein the charged particles of each substance form respective bands, the widths of the bands being successively reduced, and each of the bands overlapping each other during at least a portion of an electric field sequence, thereby facilitating the reaction between the substances.

38. 38. The method of claim 37, wherein in step (ii), the electric field sequence is repeated multiple times, preferably at least 30 times, more preferably at least 50 times, and even more preferably at least 100 times.

39. 39. The method of claim 38, wherein in step (ii), the electric field sequence may be repeated until a predetermined amount of one of the at least two substances has reacted, the predetermined amount being preferably 30%, 50%, 70%, 80%, 90%, 99% or 100%.

40. 40. The method of claim 38 or 39, wherein the cycle time is from 0.1 to 100 seconds, preferably from 0.1 to 10 seconds, more preferably from 1 to 5 seconds.

41. 41. The method of any one of claims 38 to 40, wherein the first and second electric field gradients and the duration of each cycle are configured to cause the charged particles to oscillate along the major axis of the chamber, the amplitude of the oscillation being in the range of less than 10 mm, preferably less than 3 mm, more preferably less than 2 mm.

42. 42. A method according to any one of claims 37 to 41, wherein the parameters of the electric field sequence and the respective mobilities of the charged particles of each substance are such that the respective bands of each substance cross each other, preferably repeatedly, in the chamber during step b.

43. After step (ii), 43. The method of any one of claims 37 to 42, further comprising: (iii) separating the charged particles according to their type by performing electrophoresis on the contents of the reaction chamber, wherein reaction products are separated from any remaining amounts of the at least two substances.

44. After step (ii) or step (iii), 44. The method of any one of claims 37 to 43, further comprising (iv) extracting the reaction product, preferably by applying an electric field configured to move charged particles of the reaction product to a port through which the reaction product is removed.

45. 1. A method for detecting an analyte by a binding protein that specifically binds to said analyte, said method comprising: (i) providing charged particles of the analyte and the binding protein; (ii) focusing the charged particles in a chamber using the method of any one of claims 1 to 30, wherein the charged particles of each of the analyte and the binding protein form respective bands, the widths of the bands being successively reduced, and each of the bands overlapping each other during at least a portion of an electric field sequence, thereby promoting a reaction between the analyte and the binding protein.

46. 37. A reactor for carrying out a reaction between at least two substances, the reactor comprising an apparatus for focusing charged particles according to any one of claims 31 to 36, wherein in use charged particles of each substance form respective bands in a chamber, the widths of the bands being successively reduced and each of the bands overlapping each other during at least part of an electric field sequence, thereby facilitating the reaction between the substances.

47. 47. The reactor of claim 46, wherein the controller is further programmed to control the electric field generator to carry out the method of any one of claims 37 to 45.

48. 48. The reactor of claim 46 or 47, further comprising a separation channel fluidly connected to the chamber for performing electrophoresis in the separation channel, wherein the electric field generator is further adapted to apply an electric field along the separation channel, and the controller is further configured to control the electric field generator to apply a particle separation electric field along the separation channel.

49. 49. The reactor of claim 48, further comprising an extraction port for extracting the separated band of particles from the separation channel.

50. 50. The reactor of claim 48 or 49, wherein the separation channel is connected to an outlet port of the chamber or the separation channel is continuous with the chamber and aligned along the major axis of the chamber.

51. 51. A reactor according to any one of claims 46 to 50, further comprising a detector adapted to detect separated bands of charged particles in the separation channel, said detector preferably adapted to image said bands.

52. 1. A method for separating at least two substances, comprising: (I) providing charged particles of said at least two materials; (II) focusing the charged particles in a chamber containing a sieving medium using the method of any one of claims 1 to 30, whereby the width of the group of charged particles is reduced; (III) performing electrophoresis on the group of charged particles along a separation channel containing a sieving medium, the separation channel being continuous with the chamber and aligned with the major axis of the chamber.

53. 53. The method of claim 52, wherein in step (II), the electric field sequence is repeated multiple times, preferably at least 10 times, more preferably at least 30 times, and even more preferably at least 50 times.

54. 54. A method according to claim 52 or 53, wherein the electric field sequence is repeated until the width of the group of charged particles reaches a predetermined threshold, the predetermined threshold being preferably 10 mm or less, more preferably 1 mm or less, even more preferably 0.1 mm or less.

55. 55. A method according to claim 53 or 54, wherein the cycle time is from 0.1 to 10 seconds, preferably from 0.1 to 5 seconds, more preferably from 1 to 3 seconds, and most preferably from 1.5 to 2.5 seconds.

56. 56. The method of any one of claims 53 to 55, wherein the first and second electric field gradients and the duration of each cycle are configured to cause the charged particles to oscillate along the major axis of the chamber, the amplitude of the oscillation being in the range 0.1 to 10 mm, preferably 0.5 to 5 mm, more preferably 1 to 3 mm.

57. In step (III), the electrophoresis method comprises: III') applying a particle separation electric field along the separation channel, the particle separation electric field having a field profile that causes the charged particles to move relative to the sieving medium; III'') The method of any one of claims 52 to 56, wherein the method is performed by varying the applied particle separation electric field to adjust the field profile for the separation channel, thereby separating the charged particles into respective bands of each of the at least two materials under the combined influence of electric forces due to the electric field and hydrodynamic forces due to the sieving medium.

58. 58. The method of claim 57, wherein the field profile of the particle separation electric field in step (III) has an electric field gradient, and the spatial rate of change of the electric field magnitude with distance along the separation channel is less than that of the first and second electric field gradients in step (II).

59. 37. An electrophoresis device for separating charged particles, comprising: an apparatus for focusing charged particles according to any one of claims 31 to 36; and a separation channel continuous with a chamber and aligned along the major axis of the chamber, wherein an electric field generator is further adapted to apply an electric field along the separation channel, and a control device is further configured to control the electric field generator to apply a particle separation electric field along the separation channel.

60. 60. An electrophoresis apparatus according to claim 59, wherein the control device is further programmed to control the electric field generator to carry out the method of any one of claims 52 to 58.

61. 61. An electrophoresis device according to claim 59 or 60, further comprising an extraction port for extracting separated bands of particles from the separation channel.

62. 62. An electrophoresis apparatus according to any one of claims 59 to 61, further comprising a detector adapted to detect separated bands of charged particles in the separation channel, the detector preferably being adapted to image the bands.

63. 1. A method for separating at least two substances, comprising: (I) providing a sample containing the at least two materials to provide charged particles of the at least two materials; (II) collecting the charged particles in a chamber containing a sieving medium by applying a particle collection electric field gradient along at least a portion of a major axis of the chamber, wherein the charged particles move under the influence of the electric field gradient to form at least one group of charged particles, the leading edge of the group moving slower than the trailing edge of the group due to a smaller electric field magnitude acting on the particles at the leading edge, thereby reducing the width of the group of charged particles; (III) performing electrophoresis on the group of charged particles along a separation channel containing a sieving medium, the separation channel being continuous with the chamber and aligned with the major axis of the chamber.

64. 64. The method of claim 63, wherein the particle collection electric field gradient is static.

65. 65. The method of claim 63 or 64, wherein the particle collection electric field gradient may include a zero-crossing point within a portion of the major axis of the chamber, thereby causing at least one group of the charged particles to move toward the zero-crossing point.

66. In step (III), the electrophoresis method comprises: III') applying a particle separation electric field along the separation channel, the particle separation electric field having a field profile that causes the charged particles to move relative to the sieving medium; III'') A method according to any one of claims 63 to 65, performed by varying the applied particle separation electric field to adjust the field profile for the separation channel, thereby separating the charged particles into respective bands of each of the at least two materials under the combined influence of electric forces due to the electric field and hydrodynamic forces due to the sieving medium.

67. 67. The method of claim 66, wherein the field profile of the particle separation electric field in step (III) has an electric field gradient, and the spatial rate of change of the electric field magnitude with distance along the separation channel is less than that of the particle collection electric field gradient in step (II).

68. 68. The method of claim 66 or 67, wherein the electric field is changed individually or continuously from the particle collection electric field gradient to a particle separation electric field profile between steps (II) and (III), preferably while being moved from the chamber to the separation channel.

69. 1. An electrophoresis device for separating charged particles of at least two substances, comprising: a chamber into which, in use, charged particles are injected into a sieving medium, said chamber having a major axis; a separation channel contiguous with the chamber and aligned with the major axis of the chamber; an electric field generator configured to apply an electric field along at least a first portion of the major axis of the chamber and along the separation channel; a control device configured to control the electric field generator and programmed to first apply a particle collection electric field gradient along at least a portion of a major axis of the chamber, so that, in use, the charged particles move under the influence of the electric field gradient to form at least one group of charged particles, a leading edge of the group moving slower than a trailing edge of the group due to a smaller electric field magnitude acting on the particles at the leading edge, thereby reducing a width of the group of charged particles, and then apply a particle separation electric field along the separation channel, so that electrophoresis is performed on the group of charged particles.

70. 70. An electrophoresis apparatus according to claim 69, wherein the controller is further configured to control the electric field generator to carry out the method of any one of claims 63 to 68.

71. 71. An electrophoresis apparatus according to claim 69 or 70, wherein the electric field generator comprises an array of electrodes disposed along at least the first portion of the major axis of the chamber and along the separation channel.

72. 72. An electrophoresis apparatus according to any one of claims 69 to 71, wherein the chamber is provided with at least one input port for injecting a sample into the chamber, the sample comprising at least the charged particles of at least two substances.

73. 73. An electrophoresis device according to any one of claims 69 to 72, wherein the separation channel is provided with at least one extraction port for extraction of bands of charged particles from the channel.

74. 74. An electrophoresis apparatus according to any one of claims 69 to 73, further comprising a detector adapted to detect bands of charged particles in the separation channel, the detector preferably being adapted to image the bands.

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