FR2847343A1 - Apparatus to separate electrically charged populations of analytes, e.g. heavy DNA chromosome molecules, has a chute defined by solid walls with a magnetic field to hold them in separate positions - Google Patents

Abstract

The apparatus to separate electrically charged populations of analytes, using electrophoresis, has a migration unit with at least one solid wall (Pr1,Pr2) to define a migration chute with a given height (h) for the sample solution to travel in a direction (Dm) through a magnetic field. The required populations are held at separate zones by a continuous or intermittent field.

Description

i

  TITLE: ELECTROPHORESIS DEVICE AND METHOD

  The present invention is, in general, relating to a device and a

electrophoresis method.

  More particularly, the electrophoresis device and method means according to the invention make it possible to separate charged analyte populations included as a mixture in a solution, and are particularly suitable for separation.

  nucleic acids such as double-stranded chromosomal DNAs.

BACKGROUND TECHNOLOGY:

  Electrophoresis is one of the most used techniques in molecular biology.

  It has notably been used for the study of the human genome (decoding of the human genome). It is indeed necessary to be able to differentiate the DNA molecules by

  their size, then study them one by one.

  The studies devoted to electrophoresis aim to increase its performances: the speed and the separating power. The interest of speed is obvious. That of separating power is a question of efficiency and reliability of separation. The development of increasingly simple and increasingly efficient systems has been going on for several years now. Decrypting genomes is one of the current goals. The techniques proposed in the prior art, however, suffer from various disadvantages, and in particular from a limitation in terms of separating power: they do not allow or can not easily separate DNA molecules whose size is of the order of a few millions of base pairs (1OMb), which remains insufficient and

  unsatisfactory for the separation of very large chromosomal DNAs.

  Electrophoresis is etymologically a setting in motion of charged particles by an electric field. If in a mixture of various particles, each of them advances in the electric field with a different speed, the electrophoresis becomes an efficient separation tool. It was Tiselius who, in the 1930s, first used this technique to separate proteins into

mixing in a solution.

  But the electrophoresis of DNA in the very simple medium that is the solution is not possible because under these conditions, the electrophoretic velocity is independent

  of mass, unlike the diffusion coefficient.

  Most current electrophoresis techniques therefore use a gel, or an equivalent such as a solution of neutral non-crosslinked polymer, a gel mimicked by a matrix of beads or more generally topological obstacles, for

  to separate the molecules and especially the nucleic acid molecules.

  The gel separations (polyacrylamide gels, agarose gels) use the sieving power of gels whose crosslinking confers a pore size distribution around a mean value which is adjusted case by case according to the populations to to separate so that this average size is less than the radius of gyration in solution of the molecules to be separated. These pores constitute very fine virtual corridors which communicate with each other so that the molecules are confined to them in creeping and their migration is impeded by topological obstacles. The gel techniques currently available are very long, and in a continuous field, can not separate nucleic acids whose size is greater than 50kb: either the usable fields are too low and the migration times are prohibitive, or it increases the field and the electrophoretic mobility becomes again (as in solution) independent of the mass; in addition, side effects of heating of the gel are induced by the Joule effect, which leads to a loss of resolution due to the increase of the diffusion or even the melting of the gel. To separate such nucleic acids on gel, it is therefore necessary to switch to a pulsed field, but the necessary analysis times remain very long, and the implementation delicate. In addition, the gels

  have the disadvantage of being non-reusable.

  Other research then led to the proposal of separation techniques

  of DNA on noncrosslinked polymer solution in capillary electrophoresis.

  The use of capillaries allows to evacuate the heat created by Joule effect, and thus not to affect the resolution by increasing the diffusion. We can

  thus use fields higher than gel, and gain speed of analysis.

  The obstacle matrix consists of a solution of neutral polymer (the molecules are entangled but not crosslinked). An example of such techniques is described in the article Barron, Sunada and Blanch (Electrophoresis 1996, 17: 744-757, "The effects of polymer properties on DNA separations by capillary electrophoresis in uncross-linked polymer solutions"). A dilute solution of the neutral polymer is placed in a non-confining capillary (a

  Capillary diameter 51 microns is used in the article Barron et al. supra).

  For small chains, the efficiency is all the greater as the solution is concentrated, but the speed is then affected. For long chains, the

diluted solution is required.

  In the end, these capillary electrophoresis techniques in non-crosslinked polymer solution do not make it possible to separate DNA populations whose size is greater than 50 kb (in the Barron et al., Article, the maximum size of analyte

separable is 23 kb).

  Other non-confining electrophoresis techniques, such as grafting a neutral molecule to the DNA strings to create an electrophoretic parachute for slowing down small molecules in comparison to larger ones, can also be mentioned. techniques that utilize the adsorption of molecules at an interface (N. Pernodet, V. Samuilov, K. Shin, J. Sokolov, M. H. Rafailovich, D. Gersappe, and B. Chu, Physical Review

  Letters 2000, 85 (26): 5651-5654, "DNA Electrophoresis on Flat Surface").

  These techniques nevertheless require very careful selection of the neutral molecules or interface coating required (an intermediate attraction coating of 2.25), and are therefore delicate and complex to master.

  and to implement for a wide variety of analyte solutions to be separated.

  In recent years, other electrophoresis techniques have been developed. These more recent techniques use micro-lithography. Instead of a sieve matrix gel or non-crosslinked polymer, they implement topological obstacles made of compact solid material, to play on the entropic changes of the molecules they induce. An example of such techniques is described in the article Jongyoon Han and Harold G. Craighead (Anal Chem 2002, 74: 394-401, "Characterization and optimization of an entropy trap for DNA separation"). In this paper, a migration cell with a corridor with alternating thin and wide regions is used. This cell thus repeatedly passes the molecules directly from a situation of absence of confinement to a situation of strong confinement. It is reported that this topological obstacle cell allows the separation of DNA molecules in the 1-200 kb range. The corridor has a length of 1.5 cm long and a width of 30 micrometers, and the

  time required for separation is about 30min.

  It is further indicated that if an attempt is made to extrapolate the operation of the system described to the chromosomal size DNA situation, the system should have a size of the order of 5 cm, would require a migration time of the order of 3.5 hours, and would likely achieve a purification of

  the DNA rather than a real separation.

  Another example of such a technique is provided in the article Masanori Ueda, Tetsuya Hayama, Yuzuru Takamura, Yasuhiro Horiike and Yoshinobu Baba (Electrophoresis 2002, 23: 2635-2641, "Investigation of the Possibility of Geometrical Electrophoresis"). The system described is a complex system, which combines a strong confinement of the molecules (capillary diameter much smaller than the Flory radius of the molecules), the presence of topological obstacles (bringing into play the elasticity of the analyte molecules), and a sieving effect on uncrosslinked polymer (polyvinylpyrrolidone stream). Thus, in the article Ueda et al. above mentioned, a periodically curved capillary of 0.6 micrometer diameter is

  used for molecules whose indicated diameter is about 4.5 micrometers.

  This system was also not tested in the presence of a mixture of analytes, but only in the presence of a single population (166 kb). His power of

separation is not established.

  There is therefore still a need for electrophoretic separation tools which would make it possible to achieve satisfactory performances of speed and separation power, for large molecules such as DNAs larger than 50 kb, and double-stranded DNAs. particularly, while being simple to manufacture, easy to implement, easy to re-use for different separations.

  SYNTHETIC DESCRIPTION OF THE INVENTION

  Faced with this problem, the Applicants have developed electrophoresis device and method means which do not have the disadvantages of the techniques of the prior art, and which, to do this, involve a particular confinement relationship. which, schematically, corresponds to an "intermediate" confinement between the situation of absence of confinement which is for example observed when the molecules are left free in solution, and the situation of strong confinement which is for example observed when the molecules are placed in a migration corridor whose size is smaller than the smallest of the average molecular radii of populations in a mixture. This intermediate containment situation can be parameterized as: Rmni, <h <6Rmax, that is, 0.2 <Tc <2, with: h the smallest dimension of the migration corridor, Rmin the smallest average molecular radii of mixed analyte populations (Gaussian paddle radius in solution), Rmax the largest of the average molecular radii of mixed analyte populations (Gaussian paddle radius in solution), and

  Tc the rate of confinement applied (Tc = 2R / h).

  Two main modes of operation can be applied to them: - a mode without electro-osmotic flow (without EOF), that is to say with walls with electrically neutral internal faces: in this case, the migration of negatively charged analytes is in the direction of the positive pole, and that of positively charged analytes is in the direction of the negative pole, and - a mode with electro-osmotic flow (with EOF), ie with walls with electrically charged internal faces negatively or positively: if the internal faces of the walls are negatively charged, the EOF goes from the positive to the negative pole, and when they are subjected to both the electric field and the EOF, the negatively charged analytes will be driven to the negative pole, if the internal faces of the walls are positively charged, the EOF goes from the negative pole to the positive pole, and when they are subjected to both the electric field and the

  the EOF, the positively charged analytes will be driven to the positive pole.

  The electrophoresis means according to the invention do not require the presence of topological obstacles. They may also be provided with means of

  adjustment so that they are re-usable for different separations.

  The means according to the invention make it possible to separate any type of charged analyte populations, and advantageously allow to separate populations of nucleic acids of high molecular weight included as a mixture in a solution (such as nucleic acids in the range of 50 to

250 kb, in particular).

  The electrophoresis means according to the present invention also have the advantage of not necessarily requiring the application of a pulsed field (nucleic acid populations in the range of 50-250 kb can be separated in a continuous field ), to be reusable, and to be able to be kept to a size of less than one centimeter, or even millimeter (the effective migration length to separate nucleic acid populations in the 50250kb range is approximately micrometer). The device according to the invention can therefore

  be manufactured in the form of a chip, such as micro- or nano-chip.

BRIEF DESCRIPTION OF THE FIGURES:

  In the present application, reference is made to the following figures: FIGS. 1A, 1B, 2A, 2B, 3A, 3B, 4, 5 show different representations

  schematic of a corridor of volume V according to the invention.

  Figures 6 and 7 show a schematic representation of a sectional view

  longitudinal view of an embodiment of a device according to the invention.

  Figure 8 shows an electroosmotic flow velocity profile in a negatively charged two-wall glass system according to the invention. Figures 9 and 10 show a schematic representation of a sectional view of a device according to the invention, the corridor is defined by two glass walls

  Pr, and Pr2 (Figure 9: longitudinal sectional view, Figure 10: top view).

  Figures 11, 12, 13 and 14 show photographs of a prototype device according to the invention. Figures 15 and 16 are graphs showing apparent mobility as a function of inter-wall height (h, also called thickness e), for a population

  of T2 DNA and for a population of lambda DNA (unmixed populations).

  FIGS. 17 and 18 are graphs showing the observed speed distributions for a solution comprising two populations of mixed analytes (T2 DNA and lambda DNA) at different inter-wall heights h (height h, also called thickness e): FIG. Left column: height (or thickness) of 1.2 microns; Figure 17 right column: height of 2.3 micrometers; Figure 18 left column: height of 5 micrometers; Figure 18 right column: height of 8 micrometers. The top graphs show the velocity distribution of the T2 DNA and mixed lambda DNA populations; the middle and bottom graphs show the velocity distribution of the T2 DNA population and that of the lambda DNA population, respectively,

  as extracted from the corresponding "mixing" graph (top graph).

  Figures 19A, 19B and 19C are a schematic representation of the inversion of velocities due to electroosmosis (the algebraic sum of the applied electric field velocity and the electro-osmotic flow velocity EOF is such that negatively charged molecules, such as nucleic acids,

migrate to the negative pole).

  Figure 20 gives a schematic representation of the mobility pt of T2 DNAs

  and lambda depending on the inter-wall height h between the glass plates.

  Figure 21 gives a schematic representation of the electrophoretic mobility (without EOF) plej in absolute value of the T2 and lambda DNAs in

  function of the inter-wall height h between the glass plates.

  Figure 22 presents the theoretical evolution of the true mobility 1I (in arbitrary unit) as a function of the inter-wall height h (continuous line = mobility of the

  lambda DNA population; dotted line = mobility of the T2 DNA population).

  Figures 23 and 24 show a schematic representation of a device according to the invention presented in longitudinal section (Figure 23: corridor of

  volume V bevel type; Figure 24 corridor of volume V in half-bevel).

  DETAILED DESCRIPTION OF THE INVENTION

  The present invention thus relates to a device and a method which are specially adapted to, and allow the electrophoretic separation of n population (s) PA1 electrically charged analytes, from a solution comprising them in mixture, with i ranging from 1 to n, and with n greater than or equal to 1, and advantageously with n greater than or equal to 2. Each population of analytes PA1 is characterized by a mean molecular radius R 1 which, in said solution, is

  different from that of the other PA1 population (s) of analytes loaded.

  The device according to the invention comprises a migration unit which comprises at least one compact solid wall defining a migration corridor in which there is a volume space V, in which an electric field is likely to reign. This corridor is intended to receive said solution of PAi populations of analytes to be separated, and to allow the migration of these PA populations, in a migration direction Dm contained in said volume V of said corridor on a

  migration length available Lndavailable.

  This corridor has a volume V which, along said available migration length Lmdiponible, or on an effective part thereof, is adapted to maintain each of PAi analyte populations intended to be received in this corridor at a rate of containment Tcj which is greater than or equal to 0.2 and lower or

  equal to 2, preferably greater than or equal to 0.2 and less than or equal to 1.7.

  The confinement rate TCj of a PA1 population at a point p of the available migration length LmJavailable being defined by the following ratio: Tci = [2 x (the average molecular radius Ri of the PA1 population)] / [the smallest dimension of the volume space V of said corridor which is perpendicular to the

  direction Dm of migration to point p].

  The degree of confinement required in accordance with the present invention represents an "intermediate" confinement between the situation of absence of confinement which is for example observed when the molecules are left free in solution, and the situation of strong confinement which is for example observed when the molecules are placed in a migration corridor whose size is smaller than the smallest of the average molecular radii of the populations in mixture. This intermediate containment situation can be parameterized as: Rmin, h 6Rnax ,, that is, 0.2 <, te <2, with: h the smallest dimension of the migration corridor, Rmin, the most small of the average molecular radii of mixed analyte populations (Gaussian paddle radius in solution), Rmax the largest of the average molecular radii of mixed analyte populations (Gaussian paddle radius in solution), and

  the rate of confinement applied (Tc = 2R / h).

  The PA1 populations of analytes must be kept in "intermediate" confinement according to the invention over a sufficiently long distance for the desired separation to occur (significantly different mean migration rates, taking into account the standard deviation at within each population); this necessary length is the efficient migration length Lmefficacious. The corridor of the cell can therefore offer the desired confinement over the entire length of migration it offers (LM available migration length available). It is nevertheless possible to choose a cell which offers an available migration length Lm unavailable slightly greater than the migration length which would have been theoretically sufficient (effective Lmefricace migration length). In this case, the corridor has the "intermediate" containment required in accordance with the invention over a portion of available migration length Lm unavailable. This part must, however, correspond to an effective part, that is to say a part at the entrance of which the PA1 populations are not separated (migration speeds are not significantly different), but at the exit of which the populations are separated (significantly different migration rates). This effective part of the corridor will therefore represent

  generally a majority part of this corridor.

  The corridor of the device according to the invention comprises at least one compact solid wall. By compact solid wall, here is meant a wall which is considered by those skilled in the art as substantially non-porous in operation, for example non-porous in contact with a solution such as a solution of analytes contained in an electrophoretic buffer for example a glass slide. This wall can not be made of a crosslinked or liquid material. The average molecular radius of a PAi population of analytes, i.e., Ri, is the mean radius of the molecular ball in the solution in which the

molecule before migration.

  The generic name of this ray is the radius of gyration, symbolized Rg. This radius Rg varies as N-0'5 when the conformation of the macromolecular ball is Gaussian, where N represents the number of segments of the molecular chain. It corresponds to the radius that this molecule exhibits under unsatisfactory solvation conditions, such as conditions of high salinity (for example, a buffered solution whose salt concentration is equal to 1 M) or low temperature (lower than room temperature). ) or theta condition. So he is

  designated, strictly speaking, by the symbol Rgo.

  Under better solvation conditions (for example a molecule placed in a solvent that does not include too many salts, such as a 10-2M solution of TBE), the radius of the molecule increases. Flory showed that in this case, the radius of the macromolecule (classically symbolized Rf) varied as N-06. The Rgo

  of a molecule is therefore slightly lower than its Rf.

  In the present invention, the molecular radius to be considered is that which actually has the molecule concerned in the solution in which it

  is located; he may therefore, depending on the circumstances, be a Rf or a Rge.

  To measure the average molecular radius of a population of analytes, one can

  proceed by any means known to those skilled in the art.

  A conventional means comprises the implementation of a microscope provided with a millimeter reticle previously calibrated by the observation of a ruler graduated micrometrically through the lens of the microscope used. The observation of the molecular balls through the reticle makes it possible to measure the average molecular radius. If desired, in order to facilitate the observation of the molecules, it is also possible to mark the molecules that are the subject of the measurement with the aid of detection markers, such as fluorescent markers for the DNA molecules (markers: fluorescein, YOYO, TOTOT OR BOBO from Molecular

Probes, Oregon, USA).

  Alternatively, the human eye can be replaced by an image acquisition and processing chain composed of a camera (for example, a Hamamatsu C2400-08 camera) and an acquisition card (for example, Matrox

  Pulsar) and an image processing software (Matrox Mil) 6.0, for example).

  Remarkably, the volume space V of said migration corridor may be devoid of a topological obstacle. It then has internal faces which,

  macroscopically, are rectilinear.

  In an embodiment especially adapted for the electrophoretic separation of n population (s) of nucleic acids PAi whose average (s) molecular (s) radius (s) (respectively) Ri is (are) between 0 , 5 and 1 micrometer inclusive terminals, from a solution comprising them in a mixture, a device according to the invention comprises a volume space V whose smallest dimension is conventionally between 1.2 and 5 micrometers, terminals included, preferably between 2 and 4 micrometers, for example 2.3 micrometers. This device has the advantage of allowing the separation of PAi populations

  DNA whose size is greater than 50kb, for example between 50 and 250 kb.

  For example, for a mixture of a population of 1 micrometer average molecular radius (such as a T2 DNA population) and a population of average molecular radius 0.5 micron (such as a lambda DNA population ), a constant height of 1.2 microns (Tclambda = 0.83, TCT2 = 1.67), 2.3 microns (TCIambda = 0.43, TcT2 = 0.9), or 5 micrometers can be chosen (TC] ambda = 0.2, TCT2 = 0.4), cf. Example 2 below. On the other hand, a height

  constant of 8 micrometers would not be suitable (TC) ambda = 0.25, but TCT2 = 0. 13).

  According to an advantageous embodiment of the invention, the electrophoresis device further comprises means for adjusting said volume V to make it possible to vary this volume V as a function of the average molecular radii Ri of the PA1 populations of charged analytes intended for to be received in said migration corridor. Thanks to these means, the same device can be used for a variety of successive electrophoretic separations; the device

  according to the invention thus has a very great adaptability.

  The prototype shown in illustration in the examples which follow thus comprises threaded rods (see FIGS. 9 and 11-14). Those skilled in the art can nevertheless of course provide any type of adjustment device which is adapted to the particular passage volume selected, to the desired adjustment accuracy, and to the desired handling facility. Examples of adjustment means include

  a translation plate or a tilt plate with micrometric adjustment.

  Said migration corridor may have a volume V chosen from the group consisting of a volume with constant section (see FIGS. 1A, 1B, 4) and a volume with

  divergent sections (see Figures 2A, 2B, 3A, 3B, 5).

  For example, said migration lane may have a volume V chosen from the group consisting of a parallelepipedal volume (FIGS. 1A, 1B), a volume of bevel type (FIGS. 2A, 2B), a volume of type demibiseau (FIGS. 3A, 3B ), and a cylindrode volume such as a cylindrical volume (Figure 4), a

conical trunk volume (Figure 5).

  According to a particular embodiment of the invention, the electrophoresis device has a volume space V which is defined by at least one compact solid wall is delimited by at least two compact solid faces FI and F2 facing one of the other, such as a volume space V which is defined by at least two compact solid walls Pr, and Pr2 facing each other (cf.

Illustrative Figures 6 and 7).

  These at least two compact solid faces FI and F2 facing one another are then separated by: a constant height h which is not zero (see FIG. 6), or by a gradient of non-zero heights Vh , which extends from the lowest height hmin to the highest height hmax, with hmin <hmax (see Figure 7), so as to define between them said volume migration corridor V, and - said height h constant is greater than or equal to the smallest of the average molecular radii (Rmin) PA1 populations intended to be received in said corridor, but less than or equal to six times the largest average molecular radii PA1 analyte populations intended to be received in said corridor (6 x Rmax), or where appropriate, - said gradient Vh is such that its lower bound hmjn is greater than or equal to said Rmjn, and its upper bound hmax is greater than or equal to 6

x Rmax.

  The "smallest dimension of the space of said corridor which is perpendicular to said migration direction Dm" is here the height h, or the height gradient h,

inter-face space.

  Said at least two faces FI and F2 facing each other may both be placed parallel to said migration direction Dm (see FIGS.

1A, 1B, 4, 6).

  It is also possible to place one of the at least two faces opposite one another FI or F2, or both faces FI and F2, so that it (s) form (s) an angle 0 non-zero with respect to said migration direction Dm (see FIGS. 2A, 2B,

3A, 3B, 5, 7).

  According to a particular embodiment of the invention, one of said at least two faces F 1 and F 2 facing each other forms a non-zero angle θ with respect to said direction Dm of migration, such that this angle 0 non-zero is less than or equal to 0max = Arcsin [6Rmax / L], with L length of the corresponding face, and the other of said at least two faces FI and F2 opposite one another is parallel to said migration direction Dm (half-taper type volume, see Figures 3A, 3B). According to another particular embodiment of the invention, said at least two faces FI and F2 facing each other each form a non-zero angle θ with respect to said migration direction Dm, respectively 01 and 02, with 01 equal to or different from 02, and these angles 01 and 02 are both less than or equal to 0max = Arcsin [3RInaX / L], with L length of the corresponding face (volume of

bevel type, cf. Figures 2A, 2B).

  The nonzero angle θ that one of said at least two faces forms with respect to said migration direction Dm may be equal to that formed by the other of said at least two faces, or it may be different. Regarding this aspect, there is therefore no

  no constraint in terms of machining and adjustment.

  The device according to the invention may further comprise means for easily feeding it in solution. It is recommended to ensure that the solution is provided in said volume space without creating gaseous bubbles within the liquid layer in the corridor. Advantageously, the device according to the invention may therefore comprise a supply duct intended to supply said corridor of volume V in solution of PAi populations of analytes to be separated. This conduit may for example take the form of a capillary or a chimney (symbolically illustrated by a small plastic tube in Figures 12, 13, 14), or even the shape of a finish neck at the start of the

migration corridor.

  The device according to the invention can operate according to two major types of modes: a mode without electro-osmotic flow (without EOF), and a mode with flow

electro-osmotic (with EOF).

  In an operating mode without EOF, the or each of the compact solid walls of said volume corridor V has an electrically neutral or electrically insulated inner face, for example a non-ionizable internal surface, ie an internal surface which in operation, does not load significantly when wetted by the ionic solution. For example, the or each compact solid wall of said volume corridor V may be a compact solid material wall, covered on its inner face with an electrically insulating coating layer. According to the present invention, it will be preferable that said or each of the electrically neutral inner faces has an attraction 8, less than or equal to 2.0, that is to say a zero or low attraction, so as to avoid the phenomena of adhesion and adsorption of molecules, which are likely to disrupt the quality of the separation. For example, a coating having an attraction of less than or equal to 2.0, such as a neutral polymer coating, for example a coating selected from the group consisting of a PVP (polyvinylpyrrolidone) coating, a coating of PDMA (polydimethylacrylamide). It is recommended to ensure that the coating

  insulation covers the entire inner face of the wall, leaving no uninsulated area or microzone.

  In an operating mode with EOF, the or each of the compact solid walls of said volume corridor V has an internal face capable of electrically charging, or more precisely an electrically ionizable inner face (internal face which is in a material which comprises chemical groups ionizable). This or each of these inner faces is thus capable of being electrically charged according to the pH of the solution with which it is brought into contact. An inner surface capable of being negatively charged in contact with a basic solution such as a solution at pH 8.3 will be chosen more particularly. In a mode of operation with EOF, this or internal face is not covered with

electrically insulating material.

  In operation, because of the electric charges of this or these face (s), it is created against this or these negative internal faces, a liquid layer of thickness K-, which comprises an excess of positive charges (layer DebyeHtickel) . In Figure 8, is schematically represented the velocity profile of the electroosmotic flow between two negatively charged glass plates (the length K-1 has been exaggerated for the sake of clarity, but it is

  the nanometer order for a solution whose ionic strength is 10-2M).

  When the electric field is applied, this (or these) layer (s) of counter-ions move towards the negative electrode, and entails (s) with it (s) the liquid. It creates an electro-osmotic flow or EOF. It is recommended to ensure that this internal face loaded is of hydrophilic type, that is to say a surface "easily wettable by an aqueous solution". A surface is considered by those skilled in the art to be of hydrophilic type when a drop of water deposited on this surface makes a contact angle of less than 5 degrees with this surface. More precisely, a surface is considered to be of "hydrophilic type" or "wettable", when its "spreadingparameter" S is strictly greater than zero, with S = ysv-7SL-YLv where ysv represents the energy per unit area between l solid element (here, the surface of the wall) and the vapor (here, the ambient air), YSL represents the energy per unit area between the solid element (here, the surface of the wall) and the liquid (here, the aqueous solution), and 7Lv represents the energy per unit area between the

liquid and vapor.

  In the case of the separation of polyanions with EOF, it will be possible to choose a wall of negative or neutral material such as glass or mica; in the case of the separation of polycations with EOF, it will therefore be possible to choose a wall of positive or neutral material such as a wall with a surface of metal oxides (oxide of

  titanium, cerium oxide, aluminum oxide, for example).

  According to the present invention, the or each of the internal faces of said compact solid walls of said volume corridor V may have a low to zero roughness, and in particular a roughness of between 0 and 100 Angstroms, preferably between 2 and 60 Angstroms, for example of 5 to 50 Angstrom,

all terminals included.

  The or each of the compact solid walls of said volume corridor V may therefore be a glass wall (which conventionally has a roughness of 5 to 50

About Angstrom).

  The device according to the invention may further comprise means, such as a

  capillary, to collect PA1 populations of separated analytes.

  The device according to the invention may further comprise means adapted to apply an electric field within said migration space, so as to allow the induction of migration of said PA1 analyte populations along said migration direction Dm. For example, the device may comprise platinum electrodes and / or reservoirs for containing an electrophoretic buffer and / or an electric current generator (current generator

  continuous and / or alternating current).

  In a particularly remarkable manner, the device according to the invention can be miniaturized. It can especially be reduced to a size less than one centimeter, preferably a size less than one millimeter. The present invention therefore also aims at any nano- or microchip which comprises a device according to the invention. The inventors have indeed been able to demonstrate that, to separate two populations of DNA that are contained in an equimolar mixture in a solution, and whose average molecular radii R1 and R2 are 0.5 micrometre and 1 micrometer respectively (lambda DNA of about 50 kb, and 164 kb T2-D), the effective migration distance required to reach a difference of 20s between the respective migration elution peaks of the two DNA populations does not exceed 10 mm (parallel two-walled device). separated by a height of 1.2 microns). This effective migration distance can, in this example equimolar mixture of lambda DNA and T2, be reduced to 0.5 mm, by choosing an inter-wall height of 2.3 micrometers. For this example of separation of two DNA populations of about 50 and 164 kb, the device according to the invention has, in its arrangement leading to a size less than one centimeter, as in its layout leading to a size less than one millimeter, the ability to separate low PM (less than 50kb) DNA,

  as well as DNAs of high PM (size equal to or greater than 50 kb).

  It can therefore be seen that the device according to the invention has such separation performances that it can be produced in the form of a microchip or even a nanochip. Such micro- or nano-chips are particularly suitable for the separation of nucleic acid populations such as DNAs and RNAs, and especially double-stranded nucleic acids. The performances in terms of speed and separation power are in fact such that the invention makes it possible to access the electrophoretic separation of double-stranded DNA such that

chromosomal DNAs.

  The present invention also provides a method for the electrophoretic separation of n population (s) of analytes PAi electrically charged, from a solution comprising them in a mixture, with i ranging from 1 to n and with n greater than or equal to 1 , each PAi population of analytes being characterized by a mean molecular radius R 1 different from that of the other populations PA1

  analytes contained in said solution.

  The electrophoresis method according to the invention is characterized in that said solution is placed in such a way that all the PAi populations of analytes to be separated are kept at a confinement rate which is greater than or equal to 0, 2, and less than or equal to 2, and an electric field is applied, so that said populations of analytes PAi migrate from one pole to another in said migration direction Dm, each population of analytes PA1 contained in said solution acquiring a mean speed significantly different from

  that of the other populations PA1 to be separated contained in said solution.

  To do this, one can advantageously use a device according to the invention.

  In particular, when the solution is of unknown composition, that is to say when the mixed PAi populations are not previously known, it is then possible to determine the height h (the smallest dimension of said corridor at a point p in the direction of migration Dm) as a function of the smallest of the average molecular radii of the PA populations, intended to be received in said corridor (Rmin), and the largest of the average molecular radii of PAi populations intended to be received in said corridor (Rmax). The method according to the invention can then comprise the following steps: - a device or a chip according to the invention is provided, selected such that: TCi min = [2 x Rmjn] / [the smallest dimension of the volume space V of said corridor which is perpendicular to the migration line Dm], and Tcj max = [2 x Rmax)] / [the smallest dimension of the volume space V of said corridor which is perpendicular to the direction Dm of migration] are both, independently of each other, greater than or equal to 0.2 and less than or equal to 2, with Rmin being the smallest of the mean molecular radii of PA1 populations intended to be received in said corridor, and with Rmax the largest of the average molecular radii PA1 populations intended to be received in said corridor, - said solution is deposited in said device or said chip by placing it in the corridor of this device or this chip, and applies a field electr in such a way that said populations of analytes PA1 migrate from one pole to the other along said migration direction Dm. Each population of PA1 analytes contained in said solution thus acquires a mean speed that is significantly different from that of the other populations PA1 to be separated contained in said solution. People can then be allowed to migrate for the necessary time (or the respective distances) to reach

  desired elution times (or desired distance differences).

  This method has the advantage of allowing to solve high PM (separation

  DNA larger than 50kb), while remaining in a continuous field.

  It is recommended to make sure to deposit the solution by making sure that no bubbles are created. This solution can for example be provided within said migration corridor via a supply duct (chimney or neck), or directly in the volume corridor V, for example by spreading it over the first third of the migration plan and then by spreading electrophoretic buffer on

two more thirds.

  Of course, the length of said corridor of volume V must be sufficient to allow said separation. For a given choice of height or gradient of heights, this length depends on the populations to be separated, but also other operating conditions such as nature of the electric field, intensity of the electric field, nature of the buffer used. Depending on these operating conditions, the skilled person will determine case by case the minimum effective length, if necessary by means of preliminary tests. As illustrated in the examples above, it is indeed possible to separate DNAs between 50 and 250 kb in a DC field with a voltage of 1 to 30 V over a distance of less than one centimeter or even one millimeter (acquisition of a difference of 20s between the population elution peaks at Rmin-DNA lambda-and population at Rmax-DNA T2-). It can therefore, initially, be chosen to implement a device that offers an available migration distance of several centimeters in order to place itself in a very large surplus of available migration length, to then adjust and optimize the effective dimensions of the device The separation obtained after submitting the PAi populations to an "intermediate" confinement according to the present invention is in fact kept for several cm, so that an oversized length device can be constructed as a prototype. In the case of the aforementioned example, two 7.5 cm microscope slides were used in prototype; such blades therefore offer a sufficiently long migration length to allow

  any type of electrophoretic separation of nucleic acids.

  It is also recommended to ensure that the concentration of analyte populations in the solution is not too high. It has been found that too high a concentration is detrimental to the quality of the separation. The interactions between analytes hinder maximum differentiation of population velocities. Also, an overall concentration of about one tenth of the C * of the largest analyte (C * = concentration from which the molecules of the largest analyte

  would begin to touch each other) seems preferable.

  When it is desired to work in non-EOF mode, it is possible to use a device according to the invention, the corridor of which has electrically isolated and / or non-ionizable internal faces (see above). This solution of PA1 populations of mixed analytes will then be deposited in the corridor of said device on the positive or negative pole side according to the sign of the charge of the populations to be separated. In non-EOF mode, the negatively charged analyte populations migrate to the positive pole, and the analyte populations positively charged to the negative pole. If the PAi populations to be separated consist of negatively charged analytes, such as nucleic acids (DNA, in particular), we can therefore deposit the solution in the corridor on the negative pole side, the smaller molecules will come out first at the positive pole. . If the PAi populations to be separated consist of positively charged analytes, we can deposit the solution in the corridor on the positive pole side, and the

  Smaller molecules will come out first at the negative pole.

  When it is desired to work in EOF mode, it is possible to use a device according to the invention, the corridor of which has electrically charged or ionized internal faces as described above. The inventors have indeed demonstrated that, in the presence of EOF, because of the algebraic sum of the electric field and this EOF, the analyte populations migrate in the opposite direction of that observed in the absence of EOF: Negatively charged analyte populations, such as nucleic acids, migrate to the negative pole, and positively charged analyte populations migrate to the positive pole A solution of PAi populations of negatively charged analytes can therefore to be deposited in the corridor of said device on the positive pole side, while a solution of PAi populations of positively charged analytes can be deposited in said corridor on the negative pole side. If a solution feed pipe is included in the device intended to work with EOF, then this pipe (capillary or chimney, for example) will preferably be placed on the side of the most appropriate pole.

  (positive pole for negative analytes, negative pole for positive analytes).

  The device and method according to the invention are suitable for separating any type of any population of charged or chargeable molecules. Examples of molecules include double-stranded nucleic acids (especially chromosomal nucleic acids), single-stranded nucleic acids, such as double-stranded and single-stranded DNA and RNA; the proteins; ionic polymers such as ionic polysaccharides and in particular anionic polysaccharides of bacterial or plant origin such as xanthans, succinnoglycans and

carrageenans.

  Xanthans and succinoglycans can indeed be separated from a bacterial culture medium, such as for example a culture medium of Pseudomonas campestris.

  Carrageenans can be separated from crushed vegetables or plant extracts.

  These polysaccharides are particularly useful for the food industry.

  They are especially useful as thickeners in food products such as yogurt, in pharmaceutical or para-pharmaceutical products (medicines, cosmetics, hygiene products) such as toothpastes, in

products such as painting.

  The preparation of monodisperse fractions makes it possible to optimize their use when the desired property (rheofluidifier, thickener, etc.) strongly depends on

the mass.

  Remarkably, the method according to the invention can be implemented in a continuous electric field, for example, a continuous applied voltage of between 1 and 1000 V, inclusive and selected terminals according to the system to correspond to an electric field between 1 and 200 V / cm so that it allows the desired separation, while maintaining a good elimination

of the Joule effect.

  However, we can choose to apply a pulsed field.

  The present application also relates to any use of a device or method according to the invention for the separation of chromosomal DNA, or for the production of xanthanes, succinoglycans or monodisperse carrageenans, by separation from a solution of mixed container

  with other compounds, or for the separation of polyelectrolytes.

  The present invention is illustrated by the following examples, given as

  purely illustrative. They do not limit it in any way.

EXAMPLE 1: MIGRATION SPEEDS

  A prototype comprising a migration unit whose space of volume V is defined in the smallest dimension by two glass walls has been constructed (system according to the diagrams of FIGS. 1A and 6) with the aid of two blades.

  standard microscope (walls PrI and Pr2).

  Figure 9 gives a schematic representation of this prototype device in longitudinal section, and Figure 10 a schematic representation of a top view of this prototype device. Figure 11 shows a photograph. The

  glass walls are bare (that is to say not covered with an insulating coating).

  The migratory capacities of this device are tested in the presence of a solution containing a population of lambda DNA (48.5 kb, Rf = 0.5 micrometer), then in the presence of a solution containing a population of T2 DNA ( 164 kb, Rf = 1 micrometer). The solution is buffered basic (in this case Tris borate EDTA 10-2 M at pH 8.4), which makes it possible to induce negative electrical loading of the glass walls (500 mg of lambda DNA stock solution). ml / ml provided by Biolabs (ref 301-1S), and T2 DNA solution at 129, tg / ml provided by Sigma

Aldrich (ref D-4806)).

  The microscope slides (standard glass blades 7.5 cm long, Roth-Sochiel GmbH, 3, rue de la Chapelle, 67630 Lauterbourg, France) are immersed in a solution of surfactant (10% Sochiel SARL, 3, rue de la Chapelle, 67630 Lauterbourg, France) and sonicated during 15

  minutes. They are then rinsed with distilled water and oven dried at 60C.

  A good wettability of the blades is required to avoid the appearance of bubbles; therefore, care should be taken not to exceed a dry storage period of more than 2 hours. The slides can however be kept 24 hours in distilled water

  without significant alteration of their wettability.

  The lower blade is connected to two polyoxymethylene tanks by means of a silicone seal line (CAF 4, Rhône-Poulenc) to ensure

  sealing and mechanical strength.

  Two platinum electrodes (diameter 0, lmm - Société Goodfellow SARL, 229, rue Solférino, 59000 Lille, France) are inserted in the tanks to be connected to a generator (distance between electrodes = 8 cm). A standard low generator

voltage was used.

  The solution containing the analytes is brought into the interblade space by depositing a drop directly in the middle of the bottom blade, before covering this blade with the second. The solution can also be brought directly between the blades by spreading it over one third of the length of the blade, and covering the remaining two-thirds of buffer solution. Alternatively, the solution can be provided via a feed duct (or chimney) provided for this purpose. Whatever the mode of feeding chosen, it is important to ensure that the liquid is

  brought without gas bubbles forming in the liquid layer.

  Figures 12, 13 and 14 show photographs of a prototype device on which a small plastic tube placed on the upper blade symbolizes the presence of a possible feed duct: Figure 12 shows a photograph in disassembled form , Figure 13 in mounted form, and Figure 14 shows the prototype device schematically connected to a generator. In

  Figure 13, the black terminal is the positive terminal, and the red terminal the positive terminal.

  A recessed metal plate (to allow observation under the microscope) is set and held with screws to ensure the tightening and maintain the desired height interlames (also called thickness e) (piece at the top of the

  Figure 12, and appearing in the foreground in Figure 13).

  Both reservoirs are filled with buffer solution (Tris borate EDTA 102 M pH 8.4). The thickness of liquid between the blades is calculated precisely from a measurement of the intensity of the current by means of a microamperemeter placed in series (U = Ri, 1st law of Ohm; measure i, we calculate R which is

function of the thickness).

  The velocity or electrophoretic mobility (p = VDNA / E, where E denotes the electric field defined as the quotient of U by the distance between electrodes) is measured by means of a camera mounted on a microscope which records the displacement of the electrodes.

  molecules. The speed of the molecules is determined by analyzing the images of the film.

  Measurements are made for different applied voltages (20V, 30V, 40V, V and 60V). It has been verified that electrophoretic mobility (speed / field

  electric) was independent of the field.

  In this way, the speed of the T2 DNA population and that of the lambda DNA population were measured at different heights h. Figure 15 gives an illustration of the results obtained (apparent mobility of

  each population according to inter-blade height).

  It can be seen that the speeds (or mobilities) are different, and that the device of the invention thus makes it possible to migrate the 48.5 kb DNA population to a

  different speed than the 164 kb DNA population.

  FIG. 16 shows a representation of the mobilities measured as a function of the inter-slide height h (the square symbols represent the T2 DNA population, the cross symbols the lambda DNA population). It is observed that there is indeed a difference in speed between the two molecular masses for equivalent thicknesses. T2 DNA has a higher speed than lambda DNA. The phenomenon of electro-osmotic flow (EOF) reverses the order of mobilities (the strongest masses move faster). We can distinguish three regimes: - regime 1: at small thickness (smaller than the size of the chains), the measured mobility increases with the thickness and reaches a maximum, - regime II: for a thickness of the order of 2 times the size of the chains, the mobility decreases abruptly, - regime III: with great thickness (well beyond lattice of the chains), the mobility is constant, and the different sizes of chain are no longer differentiable; we are in a regime where the walls have no effect

  on the mobilities, the chains are then as in solution.

EXAMPLE 2: SEPARATION OF A MIXTURE

  The experiment described in Example 1 above is continued with a solution which comprises two populations of analytes in a mixture (48.5 kb DNA and DNA

164 kb mixed).

The prototype device described in Example 1 above is thus used for the separation of a solution comprising two populations of DNA in a mixture, namely a solution comprising, in mixture, a population of lambda DNA (48.5 kb - 1.25 μg / ml) and a population of T2 DNA (164 kb1.29 μg / ml) in a basic buffered aqueous solution (in this case Tris borate EDTA 10-2 M to

pH 8.4).

  The final concentration of lambda DNA comes from the dilution of a stock solution at 500 μg / ml provided by Biolabs, and that of T2 from a 129 μg / ml solution provided by Sigma Aldrich. For visualization and image analysis, the DNAs are labeled with YOYO-1 (Molecular probes, Eugene, Oregon, USA) at the rate of 1 dye per 50 base pairs as directed by the supplier. A mixing volume of 60 ffl is spread on the lower blade (the thickness of the

  liquid layer is then of the order of 5, tm), then covered by the upper blade.

  It is also carried out as indicated in Example 1 above.

  The upper cover is put in place and held by screws so that the filling of the side tanks with 800 μl of buffer TBE 10-2 M does not reseat the upper blade. An estimate of the liquid thickness is made from the stroke of the fine-tuning microscope knob (1 graduation = 2 jtrm) existing between the focusings of the lower face of the upper blade and the

  upper face of the lower blade.

  By varying the initial volume of mixing, we predetermine the final inter-blade height h, and thus carried out mobility measurements at different thicknesses. Results: Table 1 below presents a summary of the measured experimental values, compared to the mobilities measured for the same unmixed populations: Height h Mobility, uk Mobility IT2 Mobility jtpx Mobility JLT2 in micrometer in the in lambda alone (10-4 mixture (10-4 mixture (10-4 (10-4 cm2 / Vs) cm2 / Vs) cm2 / Vs) cm2 / Vs) 1,2 1,3 + 0,1 1,5 + 0 , 1 z 0.6 1.7

2,3 2,6 + 0,1 34 + 0,1 1,7 3,0

  l1,1 + 0,1 1,4 + 0,1 0,8 1,1 8 1,2 + O 1 1,2 + 0,1 unmeasured unmeasured Figures 17 and 18 give a graphical representation of the experimental results . Figures 17 and 18 show the velocity distributions of the lambda + T2 mixture (top graphs), the largest molecules (T2 DNA in the middle), and the smallest molecules (bottom graphs), for different confinement thicknesses. : in Figure 17, the three graphs placed in column on the left show the velocity distributions obtained with a device whose inter-blades height h is 1.2 micrometer, and the three graphs placed in column on the right the velocity distributions obtained with a device whose inter-blade height h is 2.3 micrometers; in Figure 18, the three graphs placed in column on the left show the velocity distributions obtained with a device whose inter-leaf height h is 5 micrometers, and the three graphs placed in column on the right the velocity distributions obtained with a device whose inter-blade height h is 8 micrometers. The velocity distributions of the largest molecules (T2 DNA in the middle) and the smaller molecules (bottom graphs) were obtained from the distribution of the mixture once the image processing program sorted the molecules. by size by assigning each population its speed. It is noted that in the case where the velocities of the populations are the most different (inter-leaf height h is 2.3 micrometers), the overall distribution is clearly bimodal (since the mixture

  contains two populations of different masses).

  The curves are characterized by the peak area, which distinguishes the two types of DNA populations, and the position of the peaks, which provide information on the average speed of the chains, as well as the error associated with this average.

  (width at half height divided by the root of the number of speeds measured).

  It is noted that at an inter-leaf height h of 1.2 micrometer, 2.3 micrometers and 5 micrometers, the population of lambda DNA and that of T2 DNA have, in mixture, respective average mobilities which remain significantly different, and that the device allows their separation. At an inter-blade height of 8 microns, there is no significant difference in terms of mobility. We checked that in a capillary with h = 50

  micrometers, the velocities of the two populations are identical.

  Concentration in solution analytes and entrainment effect: It is also observed that T2 DNA still has a higher mobility than lambda DNA, but lambda DNA has a greater mobility than

measured when alone.

  Since the T2 DNA retains the mobility it had when alone, we can conclude that the T2 drives the lambda with it. The concentration of the molecules used must have an important effect on this training. It is lower than the recovery concentration c * (c * (lambda) = 10 dg / ml and c * (T2) = 2 gtg / ml) in solution and we measured that the chains occupied about 1% of the surface area. the image during confinement. The slowest molecules have no place to avoid the fastest and are therefore likely to be washed away. In addition, the increase in lambda mobility decreases when increasing the inter-leaf height h (also called thickness e). It is 2.2 for h = 1.2 pim, 1.5 for h = 2.3 pm and 1.4 for h = 5 Ftm. This confirms that the shortest chains are driven by the longest. A lower concentration must prevent this effect since within the limit of a very dilute solution, the chains must behave as if they were isolated. Note also in Figure 17 the presence of a second peak to the left of

  the histogram of T2 DNA velocity (right column of Figure 17).

  It is therefore recommended in this case to dilute the solution before migration, in order to

limit this effect.

  Interpretations of the different regimes of fusion: The observed molecules are set in motion by the electric field E which applies to them an electrophoretic velocity Vel which is opposed to the field. But as these chains are in an environment confined by two charged walls, an electro-osmotic flow (EOF: Electro-Osmotic Flow) VEoF speeds carry them in the other direction. They finally have a movement with apparent speed v

(See Figures 19A, 19B and 19C).

  The horizontal axis (migration direction Dm) is the axis Ox (unit vector ux). v is written:

V = VC1 + VEOF

  Mobility is usually a scalar value (positive if the strings are positively charged). According to the preceding equation, the apparent mobility pt is written thus: M = [<el + ILEOF We call soil mobility in solution. This mobility is not dependent on the mass of molecules. The literature contains several measures of this

  mobility and all agree that its value is about 4.10-4 cm2 / Vs.

  Measurements made on T2 DNA and X-DNA are qualitatively shaped

shown in Figure 20.

  To see the influence of the confinement on the true mobility it is necessary to remove the electro-osmotic mobility with the measured mobility. We have been able to show that the speed of the EOF is not dependent on the thickness as long as it is greater than 2iJ '. This is the case in our measurements. In the previous equation yei is negative and the other two mobilities are positive. The variation of the true mobility (electrophoretic) in absolute value is represented schematically on the

figure 21.

  Firstly, it is observed that DNA A has a greater mobility than T2 and reaches the limit value us, faster than T2. So that's what intuition lets us think. Having a smaller size than T2 DNA, the A

  feels the effects of confinement to a smaller thickness.

  Then, at high inter-plate height h, that is to say with a large thickness e (e "Rg), the molecules react in the same way with the applied electric field (this is the regime III of FIG. 16) Their mobility is Psol If the thickness is decreased, the molecules begin to feel the walls and their mobility decreases (Regimen II of Figure 16) Finally, at small height interblades h, that is to say at small thickness (e <Rg), the mobilities increase (regime I of Figure 16), and here we present an interpretation of this increase in true mobility (that is, a decrease in measured mobility) with

  arguments using scaling laws.

  Explanations in scale law of regime I We have distinguished two possible interpretations of this regime. The first takes into account the chain as a solvent impermeable object. The second interpretation considers the chain as permeable to the solvent. The difference

  results obtained lies in the mass dependence of calculated mobility.

  First interpretation The molecules in strong confinement are crushed between the walls. Each chain forms a cylinder of radius Rp and of thickness e (thickness e = height h). They

  are in contact with the walls.

  Each chain will therefore be subject to three forces. The electric force of the E field, the friction force of the chain on the liquid and the friction force of the chain on

containment walls.

  The electric force FeI of the field E which pulls the molecules towards the positive electrode is written: F d (by surface inity) = -E o ú is the dielectric constant of the medium (ú = so úr), is the potential zeta DNA and 27rR2 + 27rIRe K-1 the length of Debye. It is assumed that this force per unit area acts on the surface of the cylinder, ie The electric force is: F es ER2 (1 2e Fei - R5 P 1 (+) This electric force has a positive modulus (and E.UX are negative).

  the molecules to the positive electrode.

  The FC / L friction force of the chain on the liquid is written as: Fc / L (per unit area) = -r eL, -l o i7j is the viscosity of the liquid. In electrophoresis, the chain is considered as being in free drainage, so the liquid rubs on the whole chain (and not only on the equivalent sphere). The hydrodynamic forces are then scaled over a distance Ki. This force acts on the edge of the cylinder, that is to say on a

2RP surface e.

FC / L = -2r;> _ Rje

  This friction force is oriented in the positive direction since - (v VEoF) .Ux <0.

  Finally the chain rubs on the walls. The number of monomers N 1 in contact with the walls can be expressed by Ni = N a / e (N is the number of monomers per chain and the size of a monomer). Thus the friction force on the walls is written: FC /, s = -F = = -r1N-te e o i7] is a viscosity coefficient characterizing the friction of a monomer on the walls. This second friction force is oriented as for it in the direction

negative (-v, U,> 0).

  The balance of forces is written F ('IL ± FF /.+ Fd = 0e _ -; - 1 + 11 We have measured the pt mobility of the chains v = Eu E. The electroosmotic mobility is written

PEOF> O

  o; s is the zeta potential of the walls. The mobility in solution is written Issoi = e (<or is the zeta potential of the DNA molecules, taking ris = N3 / 4a (Ga112 for a Gaussian string, we get: / I ± \ '"i = p bOF + l- \ (-) (1 + \ (-)) This force balance is now expressed as a function of z- = N'12 a / e, and is written as:

3/2 <3/2

  ts 0T <'(I + 7T) + IlEYOF + 7r 1/2 (\) T / 1 0 2 k) a Assuming that q7, -1l and that K - a, the preceding relation is written: ILE) OF + / 1-sc) l (l + /) l + Nt / 2 When Tc tends to 0 (that is to say e tending to infinity), the chains are in solution, and, u = / EOF + ys . In addition, the above equation accounts for the decrease in measured mobility when decreasing the thickness between the walls. Finally, this decrease is dependent on the mass (by N and by T ^) which confirms the observation of the different mobilities for two chains of different size. For -r, "1 (that is to say, small thickness), the calculated mobility becomes very large (Figure 22): at small thickness, u diverges.This theory will no longer be valid for confinement rates higher than 10, since when TC tends towards NI12 (the thickness is equal to the size of the monomers), the mobility of the chain should be zero even with the EOF.The chain would be blocked by the walls.

  no experiments with such high confinement rates.

  FIG. 22 represents this theoretical variation of the measured mobility pt as a function of the thickness e. We chose IIEOF + Jtsol = 1 in arbitrary unit on

this representation.

  Second interpretation We always consider the three preceding forces. But in this interpretation, the electric force acts on each monomer: it no longer acts on the surface of the cylinder. This force is written: F = NqE

o q is the charge of a monomer.

  The friction force of the chain on the walls remains unchanged: e The friction force of the chain on the solvent no longer acts on the cylinder surface but on each monomer (solvent-permeable chain) and is written: FC / By the same procedure as for the first interpretation, the measured mobility is obtained: / isoI + / 'EOF (i (71-) a / = sol = q with La mass dependence is indirect.The chains only undergo these forces if they are confined.The confinement rate of DNA A and T2 DNA is not the same for a given thickness.They do not follow this mobility. At the same thickness, we have the same problem as in the previous interpretation.This interpretation is valid only for confinement rates of less than 10. The variation of the mobility as a function of the thickness is the same.

pace in both cases.

  Finally, the relevance of one or the other of its interpretations is difficult to define. The choice is between (i) solvent chain permeability and low mass dependence, and (ii) solvent chain impermeability and high mass dependence. The measurements show an important mass dependence. However, for electrophoresis, the chains are still considered

free drainage.

  Diet It The diet It is difficult to explain. We could first think that the walls play the same role as for diffusion and decrease the electrophoretic mobility (thus increase the measured mobility). But this argument is not appropriate because the chain should be considered as being in non-drainage

  free, which is not the case in electrophoresis.

  At the thicknesses corresponding to this regime II, the radius of gyration is smaller than the thickness, but Rg is only an average of their radius. The chains fluctuate and thus have the possibility of exploring the space beyond the sphere of radius Rg and approaching one of the walls. In addition, for the thicknesses corresponding to this regime, the molecules have the possibility of moving on either side of the median plane of the two walls. They can then rub on the walls that are all of

  even pretty close. This remains difficult to quantify.

  RélZime III: The plateau observed when the confinement thickness is greater than the size of the chains, is easily interpretable. The walls no longer influence the electrophoretic mobility and the chains are in solution. The true mobility (in absolute value) is therefore of the order of 4 10 -4 cm 2 / Vs, which enables us to evaluate the electro-osmotic mobility at approximately 10-4 cm 2 / Vs. Glass Surfaces: We observed the roughness of the glass walls using an AFM (AFM atomic force microscope or "atomic force microscope"). The images obtained show a very low roughness (peaks of 5 to 50). Compared to the size of the chain, the surface is rather smooth. The separation obtained can not be

due to topological obstacles.

  Calculation of the bounds of effective heights hmin - hmax: In the light of the experiments carried out, it thus appears that when the populations of analytes present in the same solution are placed in a confinement of a level "intermediate" between the situation "free in solution "and the situation" very confined with molecular compression ", it is possible to separate these populations quite satisfactorily. This "intermediate" confinement situation can be pictorially described as a situation where: - the analyte molecules are not compressed by the walls of the migration corridor (their mean molecular radius is not crushed): they are not forced to a constant friction on these walls, they are not subjected to forces putting stake their elasticity, and o - the molecules of analytes are not for all that left free as would be in a solution: their respective mobilities are

  indeed affected by the presence of walls in their immediate vicinity.

  It was then determined that the useful height limits (or effective heights) of this "intermediate" containment situation can be expressed in terms of

  function of the average molecular radii of the populations present.

  Thus, if we consider a solution comprising 2 populations PA, and PA2 of charged analytes whose average molecular radius in this solution is R1 and R2, respectively, with R1 <R2, the inter-blade height effective for a confinement " intermediate "according to the present invention is between R1 and six times R2: R1 h 6 R2. This relation can be generalized to a solution comprising n populations PAi with i ranging from i to n, and n greater than or equal to 2; in this case, it is sufficient to determine the most and the largest of the mean molecular rays in the presence, to calculate the limits of the range of effective heights hmin (fiae = Rmin and hmax (effective) = 6Rmax. to be, if desired, over-sized and to have a himin (available) very slightly less than the hmin (effective), and a lhmax (available) greater than hmax (effective), since the separation acquired during the hmin range (effective) -hmax (effective) is preserved in

output after hmax, (eff-icace).

  More generally, the "intermediate" confinement relation according to the invention can thus be expressed in the form 0.2 <Te <2, where Te is the degree of confinement undergone by the analytes (Te is the ratio between the molecular diameter

  medium and available available height to molecules: Tec 2R / h).

  Calculation of the efficient migration efficiency Lm effective: In the presence of a solution comprising in mixture n PAi analyte populations, it is therefore possible to calculate the migration distance necessary to obtain a

  determined speed difference.

  For example, in the case of the present example, with a constant interblade height of 2.3 micrometers, we observe that the average speed of the lambda DNA population is 19 micrometers per second, and that of the population of DNA 12 is 25 micrometers per second. If it is desired to obtain an elution time difference of 20s between the populations, then an effective migration length Lm (effective) of 20 / (1-19 - 1/25) = 1583 microns must be available. The prototype device used which offers an available length of

  7.5 cm migration is therefore very oversized.

EXAMPLE 3: BEVEL SYSTEM

  As shown in Example 2 above, there is, in accordance with the present invention, a gradient of effective heights to ensure containment

"intermediate" required.

  When the solution comprises several populations of analytes to be separated, and in particular more than two populations of analytes to be separated, it is therefore advantageous to configure the device so that it does not offer a single constant height h, but a gradient of heights that runs through the hmin - hmax range. The device is then placed in a configuration of divergent sections such as those

  schematically shown in Figures 2A, 2B, 3A, 3B and 5.

  Continuing with examples 1 and 2 presented above, it is therefore possible, for example, to place the upper blade in an inclined position at an angle θ relative to the migration direction (the horizontal direction), which leads to a demibisel device. as shown diagrammatically in FIG. 24. We then have 0 = Arcsin (6Rmax / Lm). With a 7.5 cm microscope slide, you get a 0 angle

7.6 mrad.

  It is also possible to incline the two blades by an angle θ relative to the direction of migration, which then leads to a bevelled device as represented

  schematically in Figure 23. We then have O = Arcsin (3Rmax / Lm).

  These relationships between angle 0 and length reciprocally make it possible to calculate the effective migration length Lm (minimum length required) for a given angle θ.

Claims (25)

  1. A device adapted for the electrophoretic separation of n PA1 population (s) electrically charged analytes, from a solution comprising them in a mixture, with i ranging from l to n and with n greater than or equal to 1, each population of analytes being characterized by an average molecular radius R 1 which, in said solution, is different from that of the other populations PA 1 of charged analytes, characterized in that it comprises a migration unit comprising at least one solid wall compact Pr defining a migration corridor in which there is a space of volume V, in which is likely to reign an electric field, said corridor being intended to receive said solution of populations of analytes, and to allow the migration of these analyte populations in a migration direction Dm contained in said volume V of said corridor over an available migration length Lmavailable, and in that said corridor has a flight ume V which, throughout said available migration length Lmavailable, or on an effective portion thereof, is adapted to maintain each of PA analyte populations; intended to be received in this corridor at a confinement rate Tc; which is greater than or equal to 0.2 and less than or equal to 2, the confinement rate Tci of a population PA1 at a point p of the direction Dm of migration being defined by the following ratio: ce = [2 x (the mean molecular radius Ri of the population PAf)] / [the smallest dimension of the volume space V of said corridor which is perpendicular to the   direction Dm of migration to point p].   2. Device according to claim 1, characterized in that the volume space V   of this migration corridor is free of topological obstacles.   3. Device according to claim 1 or 2, which is specially adapted for the electrophoretic separation of n population (s) PAi nucleic acids whose (s) average (s) molecular (s) (respectively) Ri is (are) between 0.5 and 1 micrometer, inclusive, from a solution comprising them in a mixture, characterized in that the smallest dimension of said volume space V is between 1.2 and 5 micrometers bounded included, preferably between 2 and   4 micrometers, for example 2.3 micrometers.   4. Device according to any one of claims 1 to 3, characterized in that   it further comprises means (4) for adjusting said volume V to make it possible to vary this volume V as a function of the average molecular radii R1 of the PAi populations of charged analytes intended to be received in said migration corridor.   5. Device according to any one of claims 1 to 4, characterized in that   that said migration corridor has a volume V chosen from the group   consisting of a volume with constant section and a volume with divergent sections.   6. Device according to any one of claims 1 to 5, characterized in that   that said volume space V is defined by at least two solid walls   compact Pr, and Pr2 facing one another.   7. Device according to any one of claims 1 to 6, characterized in that   that said volume space defined by at least one compact solid wall is delimited by at least two compact solid faces F 1 and F 2 opposite one another, said faces being separated by: a constant height h which is not zero, or by - a non-zero height gradient Vh, which extends from the lowest height hmin to the highest height hmax, with hmin <hmax, so as to define between them said volume migration corridor V, and in that: said constant height h is greater than or equal to the smallest of the average molecular radii (Rmin) of PA1 populations intended to be received in said corridor, but less than or equal to six times the greatest of the average molecular radii of the populations; of analytes PA1 intended to be received in said corridor (6 x Rmax), or where appropriate, - said gradient Vh is such that its lower bound hmjn is greater than or equal to said Rmin, and its upper bound hmax is greater than or equal to said Rmin; scabies 6 x Rmax.   8. Device according to claim 7, characterized in that said at least two faces FI and F2 facing each other are both parallel to said direction Dm of migration.   9. Device according to claim 7, characterized in that one of said at least two faces opposite one another FI or F2 forms a non-zero angle 0 by   relative to said migration direction Dm.   10. Device according to any one of claims 1 to 9, characterized in that   it further comprises a supply duct (3) for supplying said   corridor of volume V in solution of PA1 populations of analytes to be separated.   11. Device according to any one of claims 1 to 10, characterized in that   that the or each compact solid wall of said volume corridor V has a non-ionizable internal surface.   12. Device according to claim 11, characterized in that the or each compact solid wall of said volume passage V is a compact solid material wall, covered on its inner face with a coating layer electrically insulating.   13. Device according to claim 1 1 or 12, characterized in that said or each of said non-ionizable inner faces has a lower attraction. or equal to 2.0.   14. Device according to any one of claims 1 to 10, characterized in that   that the or each compact solid wall of said volume corridor V has a ionizable internal surface.   15. Device according to claim 14, characterized in that the or each of the internal faces of said compact solid walls of said volume lane V has a roughness between 0 and 100 Angstrom, preferably between   and 60 Angstroms, for example from 5 to 50 Angstroms, all limits included.   16. Device according to claim 14 or 15, characterized in that the or each   compact solid walls of said volume corridor V is a glass wall.   Device according to one of the preceding claims, characterized   in that it further comprises at least one outlet capillary for collecting the   PA1 populations of separated analytes.   Device according to one of the preceding claims, characterized   in that it has a size less than one centimeter, preferably one less than one millimeter.   19. Nano- or micro-chip characterized in that it comprises a device according to   any of claims 1 to 18.   20. A method for the electrophoretic separation of n population (s) of analytes PA1 electrically charged, from a solution comprising them in a mixture, with i ranging from 1 to n and with n greater than or equal to 1, each PAi population of analytes being characterized by a mean molecular radius R 1 different from that of the other PA 1 populations of analytes contained in said solution, characterized in that said solution is placed in such a way that all the PA1 populations of analytes to be separated are are kept at a Tc containment rate; which is greater than or equal to 0.2, and less than or equal to 2, and an electric field is applied, so that said populations of analytes PAi migrate from one pole to the other in the said direction Dm of migration, each PA1 analyte population contained in said solution thus acquiring a mean speed significantly different from that of the other populations PA1 to be separated contained in said solution.   21. A method for the electrophoretic separation of n population (s) of analytes PA1 electrically charged, from a solution comprising them in a mixture, with i ranging from 1 to n and with n greater than or equal to 1, each population PA1 of analytes being characterized by a mean molecular radius R 1 different from that of the other populations PA 1 of analytes contained in said solution, characterized in that:   a device according to any one of claims 1 to   18, or a chip according to claim 19, selected such that: tcj min = [2 x Rmin] / [the smallest dimension of the volume space V of said corridor which is perpendicular to the migration line Dm], and Tci max = [2 x Rmax)] / [the smallest dimension of the volume space V of said corridor which is perpendicular to the migration direction Dm] are both, independently of one another, greater than or equal to equal to 0.2 and less than or equal to 2, with Rmjn representing the smallest of the average molecular radii of the PA1 populations intended to be received in said corridor, and with Rmax the largest of the average molecular radii of the PA1 populations intended to be received in said corridor, depositing said solution in said device or said chip by placing it in the corridor of this device or this chip, and applying an electric field, so that said populations of analytes PA, migrate from one pole to another followed said migration direction Dm, each PA analyte population contained in said solution thus acquiring a mean velocity significantly different from that of the other PA populations, to be separated contained in said solution.   22. The method of claim 20 or 21, characterized in that said solution is placed in a device whose passage is with nonionizable internal faces.   according to any one of claims 11 to 13, and in that   depositing said solution of PA1 populations of analytes in a mixture in the corridor of said device on the negative pole side, when these populations PAi are negatively charged analyte populations, or on the positive pole side, when these populations PA1 are populations of positively charged analytes 23. The method of claim 20 or 21, characterized in that said solution is placed in a device whose corridor is ionizable internal faces.   according to any one of claims 14 to 16, and in that   depositing said solution of PA1 populations of analytes in a mixture in the corridor of said device on the positive pole side, when these PA1 populations are negatively charged analyte populations, or on the negative pole side, when   these PAi populations are positively charged analyte populations.   24. Process according to any one of Claims 20 to 23, characterized in that   that said PAi populations of analytes are selected from the group consisting of double-stranded nucleic acids, single-stranded nucleic acids, such as double-stranded and single-stranded DNA and RNA; the proteins; ionic polymers such as ionic polysaccharides and especially anionic polysaccharides such as   xanthans, succinnoglycan and carrageenans.   25. Process according to any one of claims 20 to 24, characterized in that   that said electric field is a continuous field.