Method for separating biological molecules and cells in solution

Abstract

A method for separating a plurality of deformable objects, such as biological cells or biological supramolecules such as DNA, in a liquid medium by use of an electrophoretic technique combined with hydrodynamic forces. The deformable objects are introduced into a channel, having a flow axis and a cross section at right angles to the flow axis, with the minimum size of the cross section being less than or equal to 50 pm; A hydrodynamic flow is defined a in the channel together with the application of an electric field in the channel, making it possible to move the deformable objects in the channel according to the flow axis and to separate them along the flow axis. A device suitable for implementing this method. The electrolyte used for the electrophoretic separation may be a non-Newtonian fluid with viscoelastic properties.

Claims

1. Method for separating a plurality of deformable objects in a liquid medium, comprising: introducing the deformable objects into a single channel from an additional channel, wherein an intersection between the single channel and the additional channel defines an introduction zone, in order to obtain a homogeneous concentration of deformable objects in the introduction zone, the single channel having a flow axis and a cross section orthogonal to the flow axis, and the minimum dimension of said cross section being less than or equal to 200 m; and applying a hydrodynamic flow with a parabolic profile characteristic in said single channel together with the application of an electric field in said single channel, allowing the deformable objects to be displaced in the single channel according to the flow axis and to be separated along the flow axis, wherein the liquid medium is non-Newtonian fluid.

2. Method according to claim 1, wherein the introduction of the deformable objects is carried out in the introduction zone of the single channel, and the displacement of the deformable objects is carried out from the introduction zone to a detection zone of the single channel, the method further comprising: detecting deformable objects which arrive in the detection zone.

3. Method according to claim 1, wherein: the minimum dimension of the single channel is less than or equal to 25 m; and/or the ratio between the minimum dimension of the cross section and the dimension of the cross section in a direction orthogonal to that of the minimum dimension is less than or equal to 1/10; and/or the distance between the introduction zone and the detection zone of the single channel is from 500 m to 20 cm.

4. Method according to claim 1, wherein the liquid medium comprises uncharged polymers.

5. Method according to claim 4, wherein the concentration of uncharged polymers is greater than or equal to the concentration from which the polymers are in contact.

6. Method according to claim 4, wherein the uncharged polymers are chosen from polyvinylpyrrolidone, poly(ethylene glycol), polyacrylamide and mixtures thereof.

7. Method according to claim 1, wherein the deformable objects have a modulus of elasticity less than or equal to 10.sup.9 Pa.

8. Method according to claim 1, wherein the deformable objects are chosen from molecules such as nucleic acid molecules, supramolecular assemblies, cells and fragments of cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a schematic top view of an embodiment of a device according to the invention.

(2) FIG. 2 schematically shows the cross section A-A (enlarged) of the above device.

(3) FIG. 3 is a schematic illustration of the hydrodynamic and electrophoretic force fields in the channel during the separation.

(4) FIG. 4 shows results of the separation of DNA molecules ranging from 500 to 10,000 bp using the method of the invention, for various electric field and hydrodynamic flow conditions (see example 1 below). The x axis shows the time since the start of the migration (in minutes), and the y axis shows the light intensity received by the detector. A separation of the tested DNA population by conventional gel electrophoresis is also shown on the bottom left for comparison.

(5) FIG. 5 shows in greater detail three of the separations presented in FIG. 4.

(6) FIG. 6 again shows one of those separations (500 mbar and 30 V), as well as the images acquired for each of the intensity maxima, showing the possibility of visualisation of individual molecules.

(7) FIG. 7 is a diagram showing the distribution of the velocity of individual DNA molecules of 48,000 bp with various potential differences (shown in the figure) and a fixed pressure difference of 100 mbar (see example 2). The velocity of the molecules is indicated on the x axis in m/s, and the percentage of events is shown on the y axis.

(8) FIG. 8 is a diagram showing the spatial distribution of fluorescent DNA molecules of 48,000 bp in a channel having a height of 2 m (left-hand part) and in a channel having a height of 12 m (right-hand part), as explained in example 2. The altitude in m is shown on the y axis (an altitude of 0 corresponding to the centre of the channel) and the measured light intensity (in arbitrary units) is shown on the x axis. The position of the walls of the channel is shown by dashed lines. The results obtained with different migration conditions, namely: for the channel having a height of 2 m, potential difference/pressure difference pairs of 15.4 V/50 mbar; 23.5 V/120 mbar; 29.4 V/200 mbar; and 45 V/600 mbar; for the channel having a height of 12 m, potential difference/pressure difference pairs of 15 V/10 mbar; 59 V/50 mbar; 83 V/100 mbar; and 114 V/200 mbar, have been superposed on the two parts of the diagram.

(9) FIG. 9 shows results of the separation of DNA molecules ranging from 500 to 10,000 bp using the method of the invention, for various electric field and polyvinylpyrrolidone (PVP) concentration conditions in the medium (see example 3 below). The x axis shows the time since the start of the migration (in seconds), and the y axis shows the light intensity received by the detector. The top graph is obtained with 0.1% PVP, the middle graph with 2% PVP, and the bottom graph with 4% PVP.

(10) FIGS. 10 and 11 show the rheological behaviour of a solution comprising 2% PVP. In FIG. 10, the components of the complex modulus of elasticity in Pa (G and G) are shown on the y axis, and the response frequency of the fluid in Hz is shown on the x axis. In FIG. 11, the viscosity in Pa is shown on the y axis, and the shear rate in s.sup.1 is shown on the x axis, under steady-state conditions (see example 3).

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(11) The invention will now be described in greater detail and in a non-limiting manner in the description which follows.

(12) Unless mentioned otherwise, the concentrations indicated in the application are concentrations by mass.

(13) Referring to FIGS. 1 and 2, the device according to the invention comprises a support 16, which can be a glass slide or plate, and a structure 15 having a recess (for example a microfabricated structure), which can be made of silicon, sealed to the support 16 in a manner known per se. For example, it is possible to use techniques of film deposition, photolithography, etching (chemical or plasma) and adhesive bonding. The film deposition can be carried out by centrifugation, by thermal oxidation, by chemical or physical vapour deposition (CVD and PVD), by low-pressure CVD, by plasma-enhanced CVD, by spraying, etc.

(14) The structure 15 (with its recess) and the support 16 define a channel 1. The channel 1 has the form of a hollow cylinder of rectangular cross section. The principal axis of the cylinder is the flow axis 14 in the channel 1. Perpendicularly to the flow axis 14, the cross section of the channel 1 is defined by a height denoted h and a width denoted L. The height h corresponds to the minimum dimension of the cross section (it is also the distance between the support 16 and the bottom of the recess of the structure 15), and the width L corresponds to the dimension in the direction orthogonal to that of the height.

(15) In general, during use, the height h corresponds to the vertical, while the width L and the flow axis 14 are in the horizontal plane.

(16) The value of h can be chosen as a function of the size of the molecules or deformable objects to be separated. In general, it is necessary that the shear according to the height of the channel 1 has an effect on the molecule or the deformable object, which requires that the dimension of the molecules or deformable objects in the liquid medium used is not less than the height of the channel 1 by a factor of more than 200 or 100 or 50 or 20.

(17) At the two ends of the channel 1 there are provided a first reservoir 4 and a second reservoir 6 respectively. The reservoirs 4, 6 are provided with respective pressure control means 13a, 13b. Electrodes 10a, 10b are immersed in the reservoirs 4, 6 in order to allow an electric field to be generated in the channel 1.

(18) An additional channel 2 cuts the channel 1 (perpendicularly or not). The additional channel 2 is formed in the same manner as the channel 1. The intersection between the channel 1 and the additional channel 2 defines the introduction zone 9.

(19) The additional channel 2 is also provided with a first reservoir 3 and a second reservoir 5 at its ends. The reservoirs 3, 5 are provided with respective pressure control means 12a, 12b. Optionally, the reservoirs 3, 5 are also provided with respective electrodes 11a, 11b adapted to generate an electric field in the additional channel 2.

(20) During use, the channel 1 and the additional channel 2 are filled with a solution, which is suitable for electrophoresis. A sample of molecules or deformable objects to be analysed is introduced into the device through the first reservoir 3 of the additional channel 2. By means of a hydrodynamic flow generated in the additional channel 2 and/or by means of an electric field generated in the additional channel 2, the molecules or deformable objects to be analysed migrate along the additional channel in the direction towards the second reservoir 5, as far as the introduction zone 9.

(21) This first migration step makes it possible to obtain a homogeneous concentration of molecules or deformable objects to be analysed in the introduction zone 9 before the start of the actual separation of the molecules or deformable objects present in that zone.

(22) In a second stage, the migration is interrupted, and migration of the molecules or deformable objects to be analysed is then effected in the channel 1, along the flow axis 14, from the first reservoir 4 to the second reservoir 6. To that end, a hydrodynamic flow is generated in the channel 1 (especially by the pressure control means 13a, 13b of the first reservoir 4 and of the second reservoir 6).

(23) Conjointly, an electric field is generated in the channel 1 by means of the electrodes 11a, 11 b in the respective reservoirs 4, 6. Preferably, the electric field is suitable for applying to the molecules or deformable objects to be separated an electrostatic force which tends to displace them in the opposite direction to the hydrodynamic flow. The direction of the electric field therefore depends on the sign of the overall charge of the molecules or deformable objects to be separated.

(24) Referring to FIG. 3 (the top part of the figure being a top view and the bottom part a side view), during the separation, the electrophoretic force field 17 in the channel 1 during the separation is substantially uniform, both in the direction of the flow axis 14 (y axis) and according to the height h (z axis) and the width L (x axis) of the channel 1, except in the immediate vicinity of the walls of the channel (over a negligible characteristic length relative to the dimensions h and L).

(25) With regard to the hydrodynamic force field 18, it is uniform in the direction of the flow axis 14 (y axis) and according to the width L (x axis), except in the immediate vicinity of the walls of the channel (over a negligible characteristic length relative to the dimension L). By contrast, it is not uniform according to the height h (z axis). Generally, it has a parabolic-type profile characteristic of Poiseuille's law.

(26) The aspect ratio L/h of the channel 1 is chosen in order to obtain this configuration of the hydrodynamic force field: for that reason, the aspect ratio is generally greater than or equal to 10, or 15, or 20. The non-uniformity of the hydrodynamic force field 18 according to the height h is important for the effectiveness of the separation of the molecules or deformable objects. The uniformity thereof according to the width L makes it possible to avoid any loss of resolution, given that the detection is generally carried out, in the detection zone 8, in a given position (or over a given interval) along the flow axis and by summation of the signal along the width L.

(27) The desired hydrodynamic flow profiles (characterised especially by given flow and average speed values) are obtained by actuating the respective pressure control means 13a, 13b (and optionally 12a, 12b) so as to generate a pressure difference between one or more inlet reservoirs and one or more outlet reservoirs. For example, in order to generate the hydrodynamic flow which ensures migration of the molecules or deformable objects to be separated from the introduction zone 9 to the detection zone 8, a pressure difference between the reservoirs 3, 4, 5 and the reservoir 6 is generated. For example, considering the geometry of the channels, a pressure difference of from 0.01 to 10 bar, preferably from 0.05 to 4 bar, and more particularly preferably from 0.1 to 1 bar, allows the desired hydrodynamic flow profiles to be obtained.

(28) It is also possible to provide a system of valves to render the flows in the two channels 1, 2 independent of one another.

(29) The detection means are not shown in the drawings. They can comprise a microscope objective on the side of the support 16 opposite the channel 1, and a detector connected thereto, such as a CCD camera. The individual molecules or the individual deformable objects can thus be detected on the acquired image, and an overall intensity measurement in the detection zone 8 or a portion thereof as a function of the time can be effected.

(30) Means for analysing the measurements and presenting the data obtained can be associated with this device.

(31) The device can also be integrated into a lab-on-a-chip, comprising, for example, other channels, reservoirs and/or electrodes similar to those described above. For example, the lab-on-a-chip can comprise a chemical or biochemical reaction device coupled, downstream, to the separating device according to the invention. Accordingly, implementation of the separation method according to the invention makes it possible to analyse the products of a chemical or biochemical reaction carried out in the lab-on-a-chip.

(32) The lab-on-a-chip can also comprise means for collecting fractions corresponding to the different molecules or deformable objects which have been separated. These collecting means can be provided downstream of the detection zone 8. Alternatively, they can replace the detection zone 8, in which case the device is used solely for preparative purposes.

(33) These collecting means can be provided in combination with the chemical or biochemical reaction device mentioned above, or without it.

EXAMPLES

(34) The examples which follow illustrate the invention without limiting it.

Example 1Separation of a Population of Double-Stranded DNA Molecules

(35) In this example, the invention is carried out in order to effect the separation of a population of double-stranded DNA molecules having sizes of 500 bp, 1000 bp, 1500 bp, 2000 bp, 3000 bp, 4000 bp, 5000 bp, 6000 bp, 8000 bp and 10,000 bp.

(36) The population of molecules is diluted in a buffer solution comprising 180 mM of trishydroxymethylaminomethane, 180 mM of boric acid, 4 mM of ethylenediaminetetraacetic acid, 0.5 M of dithiothreitol and 2% by mass of polyvinylpyrrolidone (PVP, 360 kDa), with a total concentration of nucleic bases of 5 ng/l, which corresponds for each of the sizes under consideration to respective molar concentrations of 510.sup.12 M (500 bp), 1.310.sup.12 M (1000 bp), 4.810.sup.13 M (1500 bp), 3.610.sup.13 M (2000 bp), 4.210.sup.13 M (3000 bp), 6.310.sup.14 M (4000 bp), 5.110.sup.14 M (5000 bp), 4.210.sup.14 M (6000 bp), 210.sup.14 M (8000 bp), 1.310.sup.14 M (10,000 bp).

(37) The nucleic acids are made fluorescent by labelling with an intercalating agent (YOYO, Molecular Probes) at a rate of one probe for 4 base pairs.

(38) The device used for separating and analysing this population of molecules is as shown in FIG. 1. The channel 1 and the additional channel 2 have a width of 100 m and a height of 2 m. The introduction zone 9 and the detection zone 8 are separated by 5 mm.

(39) 200 l of buffer solution not containing biological sample are arranged in the reservoirs 3, 4, 5 and 6 of the channel 1 of the additional channel 2. A flow of solution from the reservoirs 3, 4, 5 towards the reservoir 6 is created by adjusting the pressures in order to permit saturation of the surfaces with PVP, for a period of 30 minutes. 200 l of solution containing the sample of interest are then arranged in the first reservoir 3 of the additional channel 2.

(40) A plurality of migration tests are carried out by adjusting the potential difference and the pressure difference in the channel 1 to a plurality of different values ranging from 0 to 40 V and from 200 to 700 mbar, respectively.

(41) Monitoring of the intensity in the detection zone 8 is carried out with the aid of a fluorescence microscope (100 magnification) and imaged with the aid of a CCD camera at a frequency of 5 Hz.

(42) The best resolution over the whole of the range 500 to 10,000 bp is obtained with 20 V and 200 mbar, or 30 V and 500 mbar (the latter configuration additionally ensuring a migration time of less than 8 minutes).

(43) The results are shown in FIGS. 4 to 6.

Example 2Description of the Mechanism of Separation by Monitoring Single Molecules

(44) In this example, the same experimental device as in the preceding example is used. The migration of a DNA of approximately 48,000 base pairs (phage lambda DNA) in the channel is studied. The concentration by mass of polyvinylpyrrolidone (PVP, 360 kDa) is 2%. In a first stage, a pressure difference of 100 mbar is applied, and migrations are carried out with potential differences of 0, 5, 10, 15 and 20 V. The velocity of a large number of individual fluorescent DNA molecules is measured for each of these potential differences, and the velocity distribution is studied.

(45) The results are shown in FIG. 7.

(46) It is found that the dispersion of the velocity decreases when the potential difference increases, which is evidence of an effect of spatial localisation of the DNA molecules in the channel.

(47) In a second stage, the distribution of the DNA molecules in the direction of the height of the channel is analysed. To that end, fluorescence intensity measurements are carried out in various focal planes, by means of the piezoelectric locator of the objective. The results are shown in the left-hand part of FIG. 8.

(48) Four pairs of pressure difference and potential difference conditions were used, allowing a migration velocity (according to the axis of the channel) of the DNA molecules of substantially zero to be obtained. These pairs of conditions are as follows: 15.4 V/50 mbar; 23.5 V/120 mbar; 29.4 V/200 mbar; and 45 V/600 mbar. The four corresponding light intensity curves overlap approximately, and show that the DNA molecules are concentrated in the vicinity of the walls.

(49) The experiment is then repeated with a device having a greater channel height, of 12 m. The results are shown in the right-hand part of FIG. 8. The pairs of conditions used which allow the migration speed of the molecules along the channel to be cancelled out are as follows: 15 V/10 mbar; 59 V/50 mbar; 83 V/100 mbar; and 114 V/200 mbar. It is again found that the DNA molecules are located in the vicinity of the walls of the channel.

Example 3Influence of the Nature of the Liquid Medium

(50) In this example, the same experimental device as in example 1 is used, and the separation of the same DNA population is studied. The influence of the non-Newtonian character of the liquid medium on the effectiveness of the separation is tested.

(51) To that end, migrations are carried out by varying the concentration of PVP in the buffer solution between values of 0.1%, 2% (as in example 1) and 4%.

(52) At a concentration of 0.1%, the medium is a Newtonian fluid. At concentrations of 2 and 4%, it is a non-Newtonian fluid.

(53) The pressure difference applied is 50 mbar, 150 mbar and 600 mbar for the respective PVP concentrations of 0.1%, 2% and 4%. The potential differences applied are 0, 10, 20, 23, 30 and 40 V, according to the experiments. The results are shown in FIG. 9. It is found that the separation is greatly improved when the separation medium comprises a PVP concentration greater than a certain threshold, close to the coverage concentration of 0.4% of the PVP (340 kDa), conferring non-Newtonian properties on the liquid separation medium.

(54) These non-Newtonian properties are demonstrated with dynamic mechanical analysis methods using a Couette rheometer.

(55) More precisely, the fluid is arranged between a central cylinder of radius R and a peripheral cylinder (the distance between the two cylinders being denoted R and the height of the cylinders being denoted h). The central cylinder is made to rotate with a frequency of rotation w (in s.sup.1) and the mechanical moment M induced on the peripheral cylinder is measured. The stress is determined as being =M/(2R.sup.2h). The shear rate is defined as being =R.Math.w/R.

(56) When a rotation of the cylinder that is variable over time is applied, according to a sinusoidal-type function, it is practical to represent the stress and the shear rate as a function of time by complex numbers: (t)=.sub.0(w).Math.e.sup.iwt and (t)=(w).Math.e.sup.i(wt+). This makes it possible to define a complex viscosity of the fluid (w)=(w)i.Math.(w), where (w) and (w) denote real numbers, and (t)=(w).Math.(t). There are additionally defined the moduli of elasticity G(w)=w.Math.(w) (storage modulus) and G(w)=w.Math.(w) (loss modulus).

(57) In the case of a Newtonian fluid, G(w)=0 and G(w)=w.Math..sub.0, .sub.0 being the (constant) viscosity of the fluid.

(58) By contrast, the solution comprising 2% PVP has a non-zero component G(w) which becomes dominant at high frequencies (above about 30 Hz) (FIG. 10). The viscosity, on the other hand, is constant when the shear rate varies, under steady-state conditions (FIG. 11).