Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44756—Apparatus specially adapted therefor
  • G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories

Definitions

• the present invention relates to multicapillary electrophoresis devices.
• multicapillary systems comprising a strip of several juxtaposed capillaries.
• multicapillary electrophoresis systems have been proposed in which the laser beam of excitation of the molecules is sent thereon through the walls of the capillaries, along an axis in the plane of the bar along which said capillaries are distributed. Said axis is generally perpendicular to the direction in which the capillaries extend, the fluorescence of the molecules being observed by reception means having an optical axis perpendicular to the plane of the bar of the capillaries.
• Multicapillary systems are also known in which the molecules which pass through the capillaries are excited by laser radiation which is sent, just at the outlet of the bar, in the plane of said bar and perpendicular to the direction in which the capillaries extend. .
• the fluorescence of the molecules excited by this radiation is detected in particular by means of a CCD camera which is oriented with an axis perpendicular to the plane of the capillary strip.
• H. Kambara US patent 56 67 656, variants 7 and 8
• detection takes place in the small dimension space (0.5mm) arranged between the electrophoretic separation capillary and a second capillary facing it and serving for the passage of electric current.
• Said detection space is located in a cavity filled with buffer and optically favorable.
• the electric current flows into the detection space between the two capillaries so that the samples migrate from the first capillary to the second.
• An object of the invention is therefore to propose a multicapillary electrophoresis device in which the excitation of the molecules and the detection of their fluorescence is carried out at the capillary outlet, which does not have the drawbacks of previous systems and which is particularly reliable, easy to use, and has performances allowing sequencing and genotyping at high throughput.
• the invention provides a multicapillary electrophoresis device comprising a plurality of juxtaposed capillaries, means for generating inside the capillaries an electric field which ensures electrophoretic migration, at least one source for the emission of a beam intended to excite molecules at the outlet of the capillary, means for detecting the fluorescence of the molecules excited by said beam, characterized in that it comprises means for generating another electric field, called electric confinement field, which is distributed uniformly around said capillaries and which is substantially parallel thereto, this electric field confining the electrophoretic migration field and requiring the molecules to move substantially without diverging in the axis of said capillaries.
• the device comprises at least one intermediate metal electrode directly juxtaposed to the capillaries in the vicinity of the outlet thereof, as well as a metal electrode which defines the electrophoretic field and which is arranged in line with the outlets of the capillaries, parallel to the intermediate electrode and perpendicular to the capillaries, these two electrodes imposing together, at the capillary outlet, an electric confining field;
• the potential which is applied to this intermediate electrode is at a value between those of the potentials of the electrodes imposing an electric field between the ends of the capillaries;
• It includes a detection cavity in which the capillaries are received at their end, the intermediate electrode being arranged outside this cavity;
• the wall of said cavity which is opposite the intermediate electrode has orifices ensuring electrical contact between said intermediate electrode and the electrode which is arranged in line with the capillary outlets;
• the capillaries are contiguous and only one capillary out of two is charged, the electric field at the outlet of a charged capillary being confined laterally by the electric confinement field at the outlet of the adjacent uncharged capillaries; - the potential difference applied to charged capillaries is different from that applied to uncharged capillaries;
• the capillaries are distributed in bar (s) and in the direction perpendicular to the plane of the bar (s), the electric field at the outlet of the capillaries is further confined by the geometry of a cavity in which said capillaries are received;
• a bar of capillaries is between dielectric blades which, at the outlet of the capillaries, have a recess and are spaced apart by a distance less than or equal to the external diameter of said capillaries;
• It comprises, opposite the capillaries, a plurality of orifices which are aligned with said capillaries and in which the electric fields at the outlet of the capillaries in which the molecules migrate, are channeled;
• the orifices are ends of other capillaries.
• FIG. 1 is a schematic perspective representation with cutaway of a device according to a possible embodiment of the invention.
• FIG. 2 is a schematic representation from above of the containment arrangement of the device of Figure 1;
• FIG. 3 is a schematic representation in top view illustrating the current lines according to which the molecules leaving the capillary move at the outlet of a capillary;
• FIG. 4 is a schematic representation of another possible embodiment of the invention.
• FIG. 5 is a representation similar to that of Figure 3 illustrating another possible embodiment of the invention.
• Figures 6a and 6b are schematic representations in axial and transverse section illustrating the means used to achieve confinement in the direction perpendicular to the plane of a bar of capillaries with a device of the type of that illustrated in Figure 5;
• Figures 7 and 8 are schematic representations similar to those of Figures 3 and 5 illustrating other embodiments still possible for the invention.
• the device which is illustrated in Figures let 2 comprises:
• a metal anode 5 which is arranged in the channel of the container 1 downstream from the other end of the capillary strip 2 and which extends perpendicular to the capillaries, at the level of the latter,
• an intermediate electrode 7 which is arranged in the channel of the container 1 upstream from the end of the bar 2, in the vicinity of this end and which is juxtaposed with the capillaries,
• means 8 for imposing given potential differences between the cathode 4 and the anode 5, as well as between the intermediate electrode 7 and the anode 5.
• the channel 1a is filled with a polymer buffer solution identical to that of the electrophoretic separation medium in the capillaries.
• the solution filling the channel is a viscous solution reducing the diffusion of molecules and convection movements.
• the solution filling the channel is a solution of polymers which prevents the electroosmotic flow, for example a solution in buffer of 5% of Poly (vinylpyrrolidone).
• the solution filling the channel is filled with a gel or also with a solution of heat-sensitive polymers which gels at high temperature.
• the intermediate electrode 7 is a metal electrode perpendicular to the capillaries of the bar 2 and parallel to the anode 5.
• This electrode 7 is for example a metal wire placed on the bar 2 of capillaries, transversely with respect to the latter.
• the electrode 7 may consist of two metallic wires of this type, arranged transversely on the bar 2, on either side of the latter.
• it can be constituted by a metal plate pierced with holes through which the capillaries of the bar 2 pass.
• many other embodiments may also be suitable.
• the electrodes 5 and 7 are immersed in the solution with which the channel 1a is filled.
• the potential which is applied to this intermediate electrode 7 is at a value between that of the potential applied to the anode 5 and that of the potential applied at cathode 4, so as to generate around the bar 2 of capillaries an electric field which is uniformly distributed around said bar 2 and which is substantially parallel to said capillaries.
• the intermediate electrode 7 makes it possible, with the electrode 5, to generate at the outlet of the capillaries 2a another electric field which, instead of being divergent as is generally the case, is substantially parallel to the axes of the capillaries, as illustrated by the arrows in FIG. 2 which symbolize the direction of the current lines between the electrodes 5 and 7.
• This electric field is called “confinement field” because it confines the electrophoretic migration field generated between the electrodes 4 and 5
• the fields at the outlet of the capillaries diverge strongly: they are equivalent to that of a radial point charge.
• the value of the potential of the electrode 7 is preferably chosen so that the lines along which the molecules to be analyzed move are, at the outlet of the capillary 2a, of the type of those illustrated by the arrows leaving the capillaries in the figure 3.
• the cavity 6 has a restricted cross section which contributes to the confinement of the field in the direction perpendicular to the strip 2, and further limits the intensity of the current of the confinement field and consequently the resulting Joule effect.
• this cavity 6 is defined by two plates between which the end of the capillary strip is received.
• the electrode 7 is preferably placed outside of the cavity 6, so that the gas emissions generated by said electrode do not disturb the detection zone.
• the wall of the cavity 6 which is opposite this intermediate electrode 7 then advantageously has orifices ensuring contact between said electrode 7 and the electrode 5, by means of the electrolyte solution.
• These orifices are, for example, holes with a diameter of the same order of magnitude as those of the capillaries, inserted between said capillaries.
• the container 1 is made of Plexiglas® and is 1 cm high, 1.5 cm wide and 12 cm long.
• the detection cavity 6 is arranged on the bottom of the channel la. It is rectangular in shape and has a section adjusted to that of the bar 2 of capillaries. It consists of two Teflon® plates of thickness equal to the external diameter of the capillaries used (of 360 ⁇ m), of length substantially equal to 2 cm, placed on the bottom of the main container and between which spacers are interposed. The width of these spacers (approximately equal to 0.5 cm) is adjusted so that the depth of the cavity corresponds to that of the row of capillaries. A 2 mm thick glass microscope slide pressed on the separators serves as a cover for the cavity 6.
• the capillaries 2a are coated with polyimide to their ends.
• the molecules are excited in the cavity 6 at the outlet of the capillaries 2a by a beam which is perpendicular to the axis of said capillaries and which is either in the plane in which said capillaries 2a are distributed, or perpendicular to this plane.
• the width of the bar 2 of capillaries is limited by the natural divergence of the laser beam, but the whole beam is used to excite the molecules in all the capillaries.
• an elliptical beam is used for example, the long axis of which is of a length corresponding to the width of the bar of the capillaries 2a. Only a fraction of the beam is actually used to excite the signal. The fluorescence can be detected perpendicular to the plane of the capillary strip.
• a laser beam (at a wavelength of 488 nm and emitted with a power of 10 W) burst over a width of approximately 1 cm was focused perpendicular to the axis of the capillaries, at an adjustable distance from the ends.
• the fluorescence light was collected by a camera fitted with a macro objective and a SCHOTT colored filter cutting the laser light, placed vertically over the detection zone.
• the capillaries 2a were inserted 1.5cm inside the detection cavity 6; the intermediate electrode 7 was a simple electric wire of diameter 1mm placed at 2cm from the ends (outside the cavity 6) and simply pressed against the capillaries 2a; the anode 5 was an identical wire placed opposite the capillaries 2a, outside of the detection cavity 6, 2 cm from the extremities.
• the electrodes 4, 5 and 7 were in electrical contact through the polymer solution (Hydroxylpropylcellulose at 0.5% in solution in TBE OJX buffer) which fills the cavity 6 and the capillaries 2a.
• the assembly was energized by two power supplies in series: a first high-voltage stage, between the cathode 4 at the inlet of the capillaries 2a and the electrode 5 ensured the potential difference in the capillaries 2a for electrophoretic migration (corresponding at a field of 35 to 70 N / cm); a second stage, at a lower voltage, between the intermediate electrode 7 and the anode 5, imposes the electric field in the detection cell that constitutes the cavity 6 (field of the order of 75 N / cm).
• the potential of the intermediate electrode 7 was fixed to ground. It was found that the currents coming from the capillaries 2a were confined and that the bands leaving the capillaries 2a were separated. These bands are all the more confined as the external field is intense. Other embodiments than the one just described are of course conceivable.
• the capillaries 2a are immersed in a cathode tank 3a, while the capillaries 2b are immersed in a cathode tank 3b.
• the cathode tank 3a receives an electrode 4 which, with the anode 5 immersed in the detection cavity 6, generates an electrophoretic migration field inside the analysis capillaries 2a.
• the cathode tank 3b receives an electrode 7 which is at an intermediate potential between that of the cathode 4 and that of the anode 5
• the capillaries 2a and 2b have sufficiently small thicknesses and internal diameters so that, when said capillaries 2a, 2b are arranged contiguously, the lateral widening of the field is limited by the spacing 10
• 2a, 2b is preferably less than their internal diameter.
• the capillaries 2b not loaded with samples can be of internal and external diameter or of length different from those of the analysis capillaries 2a.
• the electric field in the uncharged capillaries 2b may be different from that in the analysis capillaries 2a.
• FIGS. 6a and 6b illustrates a configuration in which the bar 2 of capillaries 2a is taken between blades 9 made of dielectric materials, said blades 9 extending beyond said capillaries.
• the distance between the blades 9 is preferably less than or equal to the external diameter of the capillaries. To this end, the blades 9 have a setback at the outlet of the capillaries.
• the current lines are then more confined.
• This confinement can be further reinforced by having capillaries 10 only opposite the loaded capillaries 2a. As illustrated in FIG. 8, this configuration makes it possible to channel into the capillaries 10 the field lines of the charged capillaries 2a and of the intermediate capillaries 2b.
• the proposed devices allow easy filling of the capillaries, the viscous polymer solution can be injected from the outlet container, once the capillaries in place.
• the samples can be loaded therein by hydraulic pressure, while in laminar flow devices, only electrokinetic injection is possible. Also, the device proposed by the invention allows greater tolerance on the alignments of the capillaries. The manufacturing of the device is simplified.

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• 1998
  • 1998-03-11 FR FR9803100A patent/FR2776072B1/fr not_active Expired - Fee Related
• 1999
  • 1999-03-11 EP EP99907681A patent/EP0981739A1/de not_active Withdrawn
  • 1999-03-11 CA CA002290135A patent/CA2290135A1/fr not_active Abandoned
  • 1999-03-11 US US09/423,925 patent/US6485626B1/en not_active Expired - Fee Related
  • 1999-03-11 AU AU27331/99A patent/AU2733199A/en not_active Abandoned
  • 1999-03-11 WO PCT/FR1999/000538 patent/WO1999046589A1/fr not_active Application Discontinuation
  • 1999-03-11 JP JP54546599A patent/JP2001525072A/ja active Pending

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