WO2000040958A1 - Milieu thermosensible pour la separation d'especes au sein d'un canal de separation - Google Patents
Milieu thermosensible pour la separation d'especes au sein d'un canal de separation Download PDFInfo
- Publication number
- WO2000040958A1 WO2000040958A1 PCT/FR1999/003304 FR9903304W WO0040958A1 WO 2000040958 A1 WO2000040958 A1 WO 2000040958A1 FR 9903304 W FR9903304 W FR 9903304W WO 0040958 A1 WO0040958 A1 WO 0040958A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- segments
- temperature
- medium according
- lcst
- medium
- Prior art date
Links
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
- G01N27/44704—Details; Accessories
- G01N27/44747—Composition of gel or of carrier mixture
Definitions
- the present invention relates to the field of separation, identification and / or analysis of particles, molecules or macromolecules, and more particularly of nucleic acids within a channel, for example in a microfluidic system. or more particularly in the context of capillary eiectrophoresis.
- Gel electrophoresis has many applications for the separation of charged particles, molecules and macromolecules, and in particular biological macromolecules such as nucleic acids (DNA, RNA, oligonucleotides), proteins, polypeptides, glycopeptides and polysaccharides.
- a particularly important application is sequencing, that is to say the reading of the genetic code of DNA. It is most often done in flat and macroscopic gels (of the order of 1 to several mm thick), composed of agarose or polyacrylamide. More recently, other gels derived from acrylamide and a whole series of acrylate or methacrylate gels have been proposed to improve this or that property of the gel.
- DNA nucleic acids
- RNA oligonucleotides
- proteins proteins
- polypeptides glycopeptides
- polysaccharides polysaccharides.
- a particularly important application is sequencing, that is to say the reading of the genetic code of DNA. It is most often done in flat and macroscopic gels (of the order of 1 to
- planar gel electrophoresis has several drawbacks.
- microfluidic system means any system in which the analysis of species is carried out by means of the transport of said species and / or of fluids within a channel or a set of channels, at least one of the dimensions is submillimetric.
- CE and microfluidic systems allow faster and more resolving separations than gels, do not require an anti-convective medium, and their properties have been widely used to carry out ion separations in liquid medium.
- the first of these limitations is electroosmosis, an overall movement of the separation medium due to the presence of charges on the walls of the capillary or canal. As this movement is often variable over time and non-uniform, it affects the reproducibility of the measurements and the resolution.
- Many methods have been proposed to combat it, such as the treatment of the surface of the capillaries by adsorption of essentially neutral species on the walls of the separation channel before the separation proper (Wiktorowicz et al., Electrophoresis, 11, 769, 1990, Tsuji et al., J. Chromatogr. 594, 317 (1992), or by treating the capillary with an acid solution (Fung et al., Anal. Chem.
- microgels capable of reducing volume at high temperature (thus leading to a dilute solution of discontinuous particles of low viscosity) and to swell at low temperature until completely occupying the separation channel (thus giving the medium a gelled character and good separation properties).
- these separation media have a viscosity which decreases more or less rapidly with temperature: it is therefore necessary to introduce them into the capillary at a temperature higher than the temperature at which the separation takes place, which may have various disadvantages.
- capillary electrophoresis devices it is very difficult to thermostate the entire capillary, and it is therefore difficult to automatically implement a polymer which would only be injectable at a temperature much higher than Room temperature.
- This medium also comprises a buffer which has the role of dissolving the block copolymer at a first temperature, and of making it transit towards the gel state at said second temperature without interrupting the separation process, and without preventing the return to a soluble state when returning to said first temperature.
- the polymers described are triblock polymers of low molecular weights (typically less than 20,000), of the polyoxyethylene-polyoxypropylene-polyoxyethylene family (POE-POP-POE) and more specifically still (POE 99 -POP 69 -POE 99 , where the indices represent the numbers of monomers of each block) (trade name "Pluronic F127).
- the two POE segments at the end of the triblock systems are water-soluble, and given the low molecular weight of the copolymer, the solutions are relatively low in viscosity up to a high concentration.
- the central central POP segment becomes more hydrophobic, and these polymers combine to form within the medium a three-dimensional organized network of predetermined structure, which gives the medium the appearance and the consistency of a gel.
- this mechanism has several drawbacks for electrophoresis. On the one hand, it gives rise to a gel state endowed with good electrophoretic separation properties only at high polymer concentrations, greater than 15 g / 100 ml or even 20g / 100ml, which leads to high friction and to long migration times.
- Triblock copolymers having the same structure, with different molecular weights, give rise to comparable physical properties and to separation performances comparable to or less than F127. Is also described in WO 98/10274 a type polymer POB 12 - 260 POE -POB 12 wherein POB means polyoxybutylene. Unlike the previous polymers, these polymers give rise to a low viscosity state at a temperature above room temperature, and to gelling by lowering of temperature in the vicinity of room temperature.
- thermoviscosifying polymers which may present in water a thermoviscosifying or thermo-thickening character.
- thermoviscosifying polymers which contain on the one hand hydrophilic parts of the prepolymer or macromonomer chain type which do not have LCST in a useful temperature range, and on the other hand parts hydrophilic of the prepolymer chain type or macromonomers which have an LCST in the said useful temperature range.
- polymers of this type cannot be used with good performance as a separation medium for electrophoresis, for several reasons. On the one hand, they exert their thermoviscosifying effect only in the presence of a relatively large amount of salt in the solution, of between about 0.4 M and several M.
- thermoviscosifying effect has a charged skeleton (L'alloret et al., Colloid. Polym. Sci., 273, 1163-1173 (1995), Hourdet , Polymer preprints, 34, 972-973 (1993), Hourdet et al., Polymer, 38, 2535-2547 (1997)).
- a usual way to confer thermosiscosifying properties on a polymer consists in constructing a molecule having portions of hydrophilic chains at any temperature which help to maintain the molecule in solution, and portions of chains with LCST which lead by raising of temperature to an attractive interaction between chains, responsible for viscosification.
- thermo-viscous media based on polymers with neutral hydrophilic skeleton.
- thermoviscosification Some examples of such media have been described in de Vos et al., Polymer, 35, 2644 (1994), but they do not give rise to thermoviscosification except in the presence of a high salt content and at temperatures of more than 80 ° C, which also makes them unusable for most possible applications as a separation matrix, and in particular for electrophoresis.
- the object of the present invention is precisely to propose a new type of separation media, the properties of which can be optimized as a function of the size of the analytes which it is desired to separate through the selection of a specific copolymer. .
- separation is intended to cover any method aiming to separate, identify or analyze, all or some of the species contained in a mixture, the said species being commonly called “analytes”.
- This separation can thus be carried out within a channel in a microfluidic system or in the context of electrophoresis.
- electrokinetic separation is intended to cover any method aiming to separate all or some of the species contained in a mixture, the said species being commonly called “analytes", by causing them to migrate within a medium under the action of an electric field, whether the field exerts its motive action on the analytes directly or indirectly, for example by means of a displacement of the medium itself, as in electrochromatography, or a displacement ancillary species such as micelles, in the case of micellar electrochromatography, or by any combination of direct and indirect actions.
- a method of electrokinetic separation according to the invention will also be considered any method of separation in which the said action of the electric field is combined with another driving action of non-electric origin.
- thermosensitive medium for the separation of species within a separation channel, said medium comprising an electrolyte in which at least one set of block copolymers is dissolved, characterized in that said block copolymers :
- the claimed separation medium therefore has the capacity to pass reversibly between a fluid state of low enough viscosity to allow its introduction into the said channel, obtained at a temperature T1, and a significantly higher state of viscosity, and in any state of cause at least twice higher, obtained at a temperature T2 at least 20 ° C higher than the temperature T1.
- said separation medium is endowed with significant separation properties for species in a range of predefined chemical composition and size.
- temperature T1 is meant in the context of the invention, either a precise temperature, or a relatively narrow temperature range, typically of the order of 10 ° width, useful for the execution of an operation particular relating to the separation process, and in particular for the introduction of the separation matrix according to the invention into the separation channel.
- the temperature T1 is between approximately 15 and 30 ° C.
- temperature T2 is meant in the context of the invention, either a precise temperature or a relatively narrow temperature range, typically of the order of 10 ° width, useful for carrying out another specific operation relating to the separation process, and in particular for the step of separation of the analytes within the channel.
- this temperature or temperature range T2 is between approximately 40 and 80 ° C.
- the LCST of a significant fraction of said segments having an LCST is between temperatures T1 and T2 and more preferably between about 20 and 50 ° C.
- the term “electrolyte” is intended to denote a condensed medium capable of conducting ions.
- this medium is an aqueous buffered medium, such as buffers based on phosphate, ths (hydroxymethyl) aminomethane (TRIS), Borate, N-tris (hydroxymethyl) methyl-3-aminopropane sulfonic acid (TAPS), histidine, lysine, etc.
- buffers usable in electrophoresis are known to those skilled in the art, and a number of them are described, for example, in “Sambrook et al.,” Molecular Cloning: a laboratory manual ", Cold Spring Harbor Lab, New York, 1989.
- electrolyte can be used in the context of the invention, in particular hydroorganic solvents such as, for example, water-acetonitrile, water-formamide or water-urea mixtures, polar organic solvents such as, also for example example, N-methylformamide.
- hydroorganic solvents such as, for example, water-acetonitrile, water-formamide or water-urea mixtures
- polar organic solvents such as, also for example example, N-methylformamide.
- the term “species” is intended to denote generally analytes. These analytes can be particles, organelles or cells, molecular or macromolecular species, and in particular biological macromolecules such as nucleic acids (DNA, RNA, oligonucleotides), nucleic acid analogs obtained by chemical synthesis or modification, proteins , polypeptides, glycopeptides and polysaccharides for which total or partial separation is desired during their electrokinetic migration within said separation medium.
- nucleic acids DNA, RNA, oligonucleotides
- nucleic acid analogs obtained by chemical synthesis or modification
- proteins proteins
- polypeptides polypeptides
- glycopeptides glycopeptides
- polysaccharides for which total or partial separation is desired during their electrokinetic migration within said separation medium.
- polymer segment or “segment” is intended to denote a set of monomers linked together covalently and having specific physicochemical properties, in particular as regards solvation.
- An example of a polymer segment within the meaning of the invention is given by a sequence of all identical monomers (homopolymer segment), or a copolymer having no significant composition correlation over lengths of more than a few monomers (segment of random copolymer type ).
- block copolymer is intended to denote a copolymer consisting of polymer segments of significantly different compositions, covalently linked together.
- the block copolymer is defined by the fact that each of the segments comprises a sufficient number of monomers to present within the electrolyte physicochemical and in particular solvation properties, comparable to those of a homopolymer of the same composition and of the same size. . He opposes the polymer statistical, in which the different types of monomers succeed one another in an essentially random manner, and locally confer on the chain global properties, different from those of the homopolymers of each of the species in question.
- the size of the homopolymer segments necessary to obtain this block character can vary depending on the types of monomers and of the electrolyte, but it is typically a few tens of atoms along the skeleton of said segment.
- a block copolymer can be constituted within the meaning of the invention, in which part or all of the segments are themselves constituted by a copolymer of statistical type, insofar as one can distinguish within the said block copolymer of zones or segments of sufficient size and difference in chemical composition to give rise from one segment to another to a significant variation in the physicochemical properties and in particular of solvation.
- non-contiguous polymer segments is intended to denote within a block polymer two segments linked together by a polymer segment of different nature.
- Block copolymers suitable for the invention have the particularity of combining in their structures, at least two types of segments.
- the first type of segment is soluble in the electrolyte used for the separation at two temperatures T1 and T2 of use of the claimed medium and preferentially does not present in said LCST electrolyte.
- soluble is intended to refer to a solubility in the electrolyte at temperatures T1 and T2.
- the second type of segment has an LCST in the electrolyte used for separation. More specifically, this type of segment is essentially soluble in said electrolyte in a range of low temperatures, and essentially insoluble in said electrolyte in a range of high temperatures. The limit between these two temperature ranges is called “minimum demixing temperature" or more commonly “LCST”.
- the copolymers used according to the invention Due to the presence of these LCST segments in their structure, the copolymers used according to the invention have the property of constituting, at low temperature, a macroscopically entangled solution. homogeneous, within which the interactions between different molecules of polymers are essentially repulsive and give rise, following a rise in temperature, to attractive interactions between some of their parts which reinforce the interactions of entanglement between chains.
- the average number of LCST segments per copolymer is greater than 2 and preferably greater than 5, and more preferably still between 8 and 40. Thanks to this large number of segments, capable of interacting attractively, a given copolymer can interact with many other copolymers, which gives the medium a high resistance during the passage of analytes.
- the length and the number of LCST segments present in the copolymers used in the media according to the invention, as well as their chemical nature, can therefore vary significantly within the scope of the invention, and it is thus possible to vary greatly the viscoelastic properties of said media according to the desired application, as will be shown more precisely in the presentation of examples of implementation.
- thermo-thickening means having either a thermo-viscosifying character or a thermo-gelling character.
- thermosetting means a medium remaining in the range or ranges of use temperature capable of flow in a macroscopic container, in a time compatible with easy handling, ie less than around 30 seconds.
- gel type state means a medium incapable of significant flow under the same conditions.
- thermoviscosifier is called a medium which does not have in the temperature range (s) of use hysteresis of their properties, or of significant dependence on its properties as a function of the rate of temperature change, for rates of temperature change usually and conveniently used in capillary electrophoresis, that is to say of the order of 1 degree per minute to ten degrees per minute.
- thermalgelling medium or so equivalent “medium giving rise to a gel-like state”
- the medium according to the invention will be of the thermoviscosifier type.
- a medium according to the invention giving rise to the temperature T2 at a gel-like state.
- all or part or a significant fraction of the LCST segments have, along their skeleton, an average number of atoms greater than 75, or have a molecular mass greater than 2500, and preferably greater at 4500.
- a significant fraction means a proportion sufficient to give rise by increase in temperature to an increase in viscosity of at least 100% or equivalent to multiplying the viscosity by a factor of 2.
- Copolymers optimized for the implementation of the invention are especially those in which all of the LCST segments represent between 2 and 25% by mass, preferably between 5 and 15% by mass and even more preferably between 8 and 15% by mass of the average total molar mass of said copolymers, or between 3 and 20% and preferably between 5 and 10% of the total composition of the copolymers in number of moles of monomers.
- the claimed separation medium can advantageously comprise a set of block copolymers, comprising a skeleton constituted by a segment or a multiplicity of segments, of a chemical nature. identical or different, and having the common characteristic of being essentially soluble or else solvated in the electrolyte at temperatures T1 and T2, to which are covalently linked a multiplicity of segments of identical or different chemical nature and having the common character of be essentially soluble or else solvated in the electrolyte at temperature T1, and essentially insoluble or poorly solvated in the electrolyte at temperature T2.
- LCST are present in a number greater than 2 and are separated from each other along an essentially linear skeleton by segments of polymer not exhibiting LCST.
- the length of these latter segments may be, in the context of the invention, or relatively uniform, or on the contrary variable, the latter variant being preferred.
- a block polymer of the comb polymer type with a skeleton essentially soluble in the electrolyte at temperatures T1 and T2, carrying a multiplicity of side links essentially soluble in the electrolyte at temperature T1 and insoluble in the electrolyte at temperature T2.
- the so-called lateral links with LCST are arranged along the skeleton in a random or non-regular manner.
- This mode of implementation is generally preferable to the opposite structure, which would include polymers consisting of a main chain consisting of LCST segments carrying hydrophilic grafts, insofar as a polymer of this second type contracts above LCST and thus cannot give rise to the continuous network of topological obstacles necessary for the implementation of the invention.
- copolymers have an average number of atoms, along a section of soluble segment comprised between two consecutive bonding points of said soluble segment with segments with LCST, greater than 210;
- copolymers have a molecular mass greater than 30,000 or a number of atoms along the main skeleton greater than 2,000 and
- copolymers have a molecular mass of between 50,000 and 3,000,000 or a number of atoms along the main skeleton of between 2,500 and 100,000 and / or
- the average number of LCST segments per chain is greater than or equal to 4, and preferably between 5 and 100.
- copolymers in which the segment or segments soluble at temperatures T1 and T2 consist of at least one polymer chosen from polyethers, polyesters such as polyglycolic acid, soluble homopolymers and random copolymers of the polyoxyalkylene type such as polyoxypropylene, polyoxybutylene, polyoxyethylene, polysaccharides, polyvinyl alcohol, polyvinylpirrolidone, polyurethanes, polyamides, polysulfonamides, polysulfoxides, polystyrenesulfonate, polyacrylamide derivatives and polymethacrylamides substituted or insoluble in said electrolyte.
- polyethers such as polyglycolic acid
- polyacrylamides and polymethacrylamides As a representative of the polyacrylamides and polymethacrylamides, mention may very particularly be made of polyacrylamide, polyacrylic acid, poly N, N-dimethylacrylamide and polyacryloylaminopropanol.
- polymers soluble in the electrolyte can be used according to the invention as soluble segments, depending on the particular application and the ease of introducing them into a block polymer of desired structure.
- the soluble segments have a high solvation in the electrolyte at the two temperatures T1 and T2.
- LCST polymers can be chosen to constitute the segments which are not contiguous to LCST within a block copolymer which can be used according to the invention, depending on the electrolyte envisaged, the temperatures T1 and T2 preferred for use and analytes to be separated.
- Many LCST polymers are known to those skilled in the art, in particular in an aqueous medium. It is thus possible to refer to the book "Polymer Handbook” Brandrupt & Immergut, John Wiley, New York.
- all or part of the LCST polymer segments are derived from one or more polymers chosen from:
- polyvinylalkyl ether such as polyvinylmethylether
- hydroxyalkylcelluloses such as hydroxyethylcellulose and methylcellulose
- etheroxides such as polyoxyalkylenes such as polyoxypropylene and polyoxybutylene
- - random and block copolymers of etheroxides such as polyoxyalkylene type copolymers having an LCST such as polyoxyethylene / polyoxypropylene and polyoxyethylene / polyoxybutylene
- alkylene homo- and co-polymers such as butylene-propylene, ethylene-propylene and ethylene-butylene and
- the copolymer used according to the invention comprises at least two non-contiguous segments with LCST property derived from the homo- or co-polymerization of monomers chosen from acrylic and methacrylic acids, N-alkyl-acrylamides or - methacrylamides such as N-ethylacrylamide, N-isopropylacrylamide, aryl-acrylamides or -methacrylamides and alkylaryl-acrylamides or -methacrylamides.
- NIPAM N-isopropylacrylamide
- NIPAM N-isopropylmethacrylamide
- N, N'-diethylacrylamide random copolymers of these monomers between them or with others.
- copolymers are particularly suitable for the invention:
- copolymers chosen from polyacrylamide / poly (N-isopropylacrylamide) can be used as copolymers in the claimed separation medium; polyvinyl alcohol / poly (N-isopropyl-acrylamide) (PVA-NIPAM), polyoxyethylene / polyoxypropylene, polyacrylamide / oxyethylene-oxypropylene copolymer, polyacrylamide / polyoxypropylene, polyacrylic acid / polyoxypropylene, polyacrylic acid / polyyric acid / polyoxypropylene copolymer -isopropylacrylamide) and polydimethylacrylamide / poly (N-isopropylacrylamide) (PDMAM-NIPAM).
- PVA-NIPAM polyvinyl alcohol / poly (N-isopropyl-acrylamide)
- PVA-NIPAM polyvinyl alcohol / poly (N-isopropyl-acrylamide)
- PVA-NIPAM polyoxyethylene / polyoxypropylene
- a medium comprising a comb copolymer carrying, along a polyacrylamide skeleton, segments with LCST essentially consisting of poly-NIPAM, and comprising along their skeleton a number of carbon atoms between 35 and 600, and whose fraction by total mass does not exceed 25%.
- polymer according to the invention which is essentially neutral. It may however be useful for certain applications, and in particular for separating lightly or uncharged analytes, or tending to associate with the polymer, to choose a polymer according to the invention deliberately charged.
- Polymers of this type can be conveniently prepared, for example by including in the segments soluble in the electrolyte at temperatures T1 and T2 a significant portion of polymerized acrylic acid.
- a copolymer of this type is more particularly described in Example 10 below. As regards more particularly the concentration of copolymer in the medium, it is generally less than 20 g / 100 ml by weight.
- the copolymers used in said medium are advantageously capable, from a concentration of the order of 5 g / 100 ml and preferably of the order of 2 g / 100 ml, by weight of making reversibly transiting said medium from a state of viscosity V1, obtained at a temperature T1, to a state of viscosity V2 at least 100% higher than V1, obtained at a temperature T2 at least 20 ° C higher than T 1 .
- the viscosity V2 is greater by at least a factor of 5 than the viscosity V1.
- the preparation of the copolymers used according to the invention can be carried out by any conventional technique of polymerization or polycondensation.
- the choice of the preparation method is generally made taking into account the desired structure for the copolymer, namely comb, linear or branched and the chemical nature of the various blocks constituting it.
- copolymers used according to the invention are obtained by: a) copolymerization of monomers essentially of the soluble type and of macromonomers essentially of the LCST type comprising at at least one of their ends a reactive function, or
- reactive function means a group allowing the molecule carrying this group to be integrated into the macromolecule during the copolymerization reaction without interrupting said copolymerization.
- an average molecular weight of the LCST segments greater than 4,000 or an average number of atoms along an LCST segment greater than 90.
- the viscosity is that obtained at a shear rate of 10 s ⁇ 1 .
- thermo-thickening character in addition to the copolymer (s) with heat-thickening character, other species which do not have these properties, such as in particular water-soluble polymers, polymers non-heat-thickening associatives, or neutral or ionic surfactants, to the provided that these adjuvants do not give rise to demixing within the separation medium or to a loss of the reversible thermo-thickening character.
- Such adjuvants can be advantageous for modulating the properties and / or the separating power of said medium. It is in particular known that the addition of certain surfactants can in certain cases strengthen the association between polymers, and therefore the thermo-thickening character.
- the present invention also relates to the use of a separation medium as defined above for the separation or the analysis of species chosen from molecular or macromolecular species, and in particular biological macromolecules such as nucleic acids.
- biological macromolecules such as nucleic acids.
- DNA, RNA, oligonucleotides nucleic acid analogs obtained by chemical synthesis or modification, proteins, polypeptides, glycopeptides and polysaccharides, organic molecules, synthetic macromolecules or particles such as mineral particles, latex, cells or organelles.
- a high viscosity allows reduce the electrohydrodynamic effects responsible for poor separation of large DNA in pulsed field and more generally reduce harmful hydrodynamic flows within a capillary or microchannel.
- the media according to the invention generally exercise their beneficial effect at best by means of a change in temperature, they are also suitable for use at constant temperature. They can thus lend themselves to introduction into the separation channel and subsequent separation of the analytes at the same temperature. This may be advantageous, in particular if there is an apparatus capable of introducing into the separation channel a medium of high viscosity, and / or not easily allowing to modify the temperature between the introduction of said medium into the channel and separation of the analytes.
- Another advantage of the high viscosities authorized by the media according to the invention is that they reduce electroosmosis (see for example Bello et al., Electrophoresis 15, 623, 1994), without the need to resort to d other modes of suppressing electroosmosis known to those skilled in the art, such as for example the use of polymers having an affinity for the capillary wall, or the grafting of neutral hydrophilic polymers on the surface. If the suppression of electroosmosis or of the interaction of the analytes with the walls effected by the media according to the invention is not sufficient for a particular application, one of the methods known from skilled in the art, and thus obtain properties even superior to those obtained with these methods used alone.
- LCST segments between 2% and 20%, and - an average molecular weight of LCST segments between 2,000 and 20,000 or an average number of atoms along a segment with LCST between 35 and 350,
- duplex DNA of size between 500 bases and several million base pairs, or particles such as latex, whole cells, whole chromosomes or organelles with a medium transiting from a viscosity V1 between 100 and 10,000 mPa m "1 s " 1 at a temperature T1 between 15 and 30 ° C to a viscosity V2 greater than V1 by a factor between 2 and 100 at a temperature T2 of the order of 40 ° C or higher and comprising between 0.1 g / 100ml and 5 g / 100ml of copolymers having:
- LCST segments between 2% and 15%, and - an average molecular mass of the LCST segments greater than 4,000 or an average number of atoms along an LCST segment greater than 90.
- these media comprise a set of copolymers chosen from:
- the use of the claimed medium also covers the variants in which the temperature is modified during the separation step, insofar as said temperature variation comprises a temperature or a range of temperatures T2 at which heat-thickening of the medium is carried out as defined above, as well as the variants in which the above cycle is repeated any number of times, preferably automatically.
- the invention is particularly advantageous in the case of automated electrokinetic separations, since it allows automatic filling of the separation channel more easily and more quickly.
- the introduction of the sample can be, in the context of the invention, carried out before, during or after the heating of a significant portion of the separation channel at temperature T2.
- the present invention also relates to capillary electrophoresis devices, including those on chips using as a separation medium a medium according to the invention. It is particularly useful in the case of so-called "on-chip” electrophoresis devices or in etched microchannels, since, in general, these devices more easily tolerate the application of high pressures for the introduction of the separation medium than the cylindrical capillaries.
- the media according to the invention and the separation methods involving these media are particularly advantageous for the applications of diagnostic, genotyping, and high throughput screening, quality control, or for detecting the presence of genetically modified organisms in a product.
- Figure 1 Evolution of the viscosity as a function of temperature for different PAM-NIPAM, PDMA-NIPAM copolymers and for a conventional polyacrylamide in solution at 5 g / 100 ml in water.
- Figures 2 Evolution of the viscosity as a function of the temperature of copolymers according to the invention
- Figures 3 Example of separation of duplex DNA fragments in the size range 100-12000 base pairs ("kb ladder", Life Technologies, Paisley, UK) in a separation medium according to the invention based on a PAM-NIPAM polymer (T15) in solution at 2 g / 100ml in TRIS-TAPS buffer at 25 ° C ( Figure 3a) and 60 ° C ( Figure 3b).
- Figures 4 Electropherograms representing the separation of restriction fragments "PhiX-174-RF DNA Hae III digest” (Pharmacia biotech) in a medium according to the invention obtained based on polymer T7, at 20 ° C ( Figure 4a) and at 50 ° C ( Figure 4b).
- Figures 5 Example of separation of duplex DNA fragments (100 bp fluorescein ruler, Bio-Rad) in a separation medium according to the invention based on T21 at two temperatures 20 ° C ( Figure 5a) and 60 ° C ( Figure 5b).
- Figures 6 Portions of electropherograms representing the separation of a sequence reaction product obtained with a medium according to the invention based on a PAM-NIPAM (T10) copolymer at 5 g / 100 ml in TRIS-TAPS 7M urea buffer at 60 ° C ( Figure 6c), PAM-NIPAM (T10) at 3 g / 100ml ( Figure 6b) and with a commercial sequencing medium (POP6 Perkin-Elmer) ( Figure 6a).
- PAM-NIPAM T10 copolymer at 5 g / 100 ml in TRIS-TAPS 7M urea buffer at 60 ° C
- PAM-NIPAM T10 at 3 g / 100ml
- Figure 6a a commercial sequencing medium
- Figures 7 Portions of electropherograms representing the separation of a sequence reaction product obtained with a medium according to the invention based on a PAM-NIPAM (T10) copolymer at 5 g / 100 ml in TRIS-TAPS 7M urea buffer at 60 ° C ( Figure 7c), PAM-NIPAM (T10) at 3 g / 100ml ( Figure 7b) and with a commercial sequencing medium (POP6 Perkin-Elmer) ( Figure 7a).
- PAM-NIPAM T10 copolymer at 5 g / 100 ml in TRIS-TAPS 7M urea buffer at 60 ° C
- PAM-NIPAM T10 at 3 g / 100ml
- Figure 7a a commercial sequencing medium
- FIG. 8 Electrophoregrams representing the separation of large duplex DNA "high Mw markers” (Life Technologies, Paisley, GB), by electrophoresis drawn from a medium according to the invention obtained based on polymer T10 at 60 ° C.
- Figures 10 Example of separation of duplex DNA fragments in the size range 50-500 base pairs (sizer 50-500 bp, Pharmacia biotech) in a separation medium according to the invention based on PVA-NIPAM at two temperatures 20 ° C ( Figure 10a) and 40 ° C ( Figure 10b).
- Figures 11 Electropherograms representing the separation of the sizer 50-500 bp, Pharmacia biotech, obtained with in a medium based on POE ( Figure 11a), based on POP-POE-POP at 25 ° C ( Figure 11 b) and at POP-POE-POP base at 45 ° C ( Figure 11 c).
- Figure 12 Bandwidths for a series of separations of single-stranded DNA fragments in denaturing medium (fluorescein 50 bp ladder,
- the radical polymerization of NIPAM is carried out in pure water, at a temperature slightly above ambient but below
- the initiator is a redox couple whose oxidant is potassium persulfate, K 2 S 2 O 8 (KPS) and the reducing agent is aminoethanethiol
- the priming reaction is:
- the AET.HCI also acts as a transfer agent, which makes it possible to control the length of the chains.
- NIPAM NIPAM
- 200 ml 20 g of NIPAM (0.18 mole) and 200 ml are introduced into a 500 ml three-necked flask surmounted by a condenser and equipped with a nitrogen inlet device. of water, the mixture is then stirred and heated to 29 ° C by a water bath. The nitrogen bubbling is started. After 45 minutes, 0.42 g of AET, HCl (0.0037 mole) previously dissolved in 20 ml of water is added, then 0.0018 moles of potassium persulfate (KPS) dissolved in a minimum amount of 'water. The mixture is kept under stirring for 3 hours. The solution is then concentrated and then lyophilized.
- KPS potassium persulfate
- the solid obtained is redissolved in 100 ml of methanol.
- the hydrochloride present is neutralized by adding 0.0037 mole of KOH (ie 0.208 g dissolved in about 25 ml of methanol) incorporated dropwise into the solution.
- KOH ie 0.208 g dissolved in about 25 ml of methanol
- the salt formed, KCI precipitates and is extracted by fiitration.
- the filtrate thus recovered is concentrated and then poured dropwise into 4 liters of ether.
- the polymer precipitates and is recovered by fiitration on a No. 4 frit.
- the solid is then dried under vacuum of a vane pump.
- the mass yield is of the order of 50%.
- the PNIPAM macromolecules synthesized have amino functions at the end of the chains, these originating from the aminoethanethiol initiator AET.HCI.
- AET.HCI aminoethanethiol initiator
- the mixture is then concentrated to 15 ml and then poured dropwise into 200 ml of ether to precipitate the polymer. It is filtered on a No. 4 frit and the solid is washed with three times 100 ml of ether and then it is dried under vacuum with the vane pump overnight.
- a poly- (NIPAM) macromonomer is thus obtained carrying an allyl function at the end of the chain, with a mass yield of the order of 70%.
- the molar masses of the macromonomers thus prepared were measured by SEC (size exclusion chromatography) under the following conditions: a: in 0.5 M aqueous LiNO 3 solution at 20 ° C using 4 Shodex OH Pack B803 to B806 25 cm columns in series, with refractometric detection and calibration of molecular weights relative to a POE standard b: in THF at 40 ° C, with ultrastyragel column, double refractometric detection and universal calibration against polystyrene samples.
- This second method of determination is more certain; because on the one hand the universal calibration overcomes the difference in flexibility between the chains of the polymer to be studied and standards, and on the other hand because THF is a better solvent for PNIPAM than water.
- the subject of this example is the preparation of copolymers according to the invention by copolymerization of PNIPAM macromonomer (s) obtained according to Example 1 with monomers of the water-soluble type.
- PNIPAM macromonomer s obtained according to Example 1 with monomers of the water-soluble type.
- a) Synthesis This copolymerization is carried out in water at room temperature.
- the initiator used is the redox couple ammonium persulfate ((NH 4 ) 2 S 2 O 8 ) [20 g / 1] - sodium metabisulfite (Na 2 S 2 O 5 ) All of the copolymers thus prepared are purified by precipitation in acetone, with the exception of the T7 copolymer, the soluble segments of which consist of dimethylacrylamide DMAM, which is purified by ultrafiltration and lyophilization.
- Ns the average number of LCST segment per polymer (mass averages), denoted Ns, is easily deduced in Table 2, using the formula:
- Ns f Mw (copolymer) / Mw (macromonomer)
- PNIPAM-10 and the monomer under consideration are introduced into a 100 ml flask as well as 30 ml of distilled water. This mixture is stirred for two hours at room temperature with nitrogen bubbling in order to remove the dissolved oxygen. The mixture is then brought to the temperature chosen for the polymerization using a thermostated bath, then the initiators are added in the form of a solution of (NH 4 ) 2 SO 8 at 20 g / 1 and of a solution of Na 2 S 2 O 5 to 5 g: L, ie respectively 0.1% and 0.03% by mole of the amount of monomers introduced. The mixture is kept under stirring and bubbling for 4 hours. Before the introduction of the initiators, then every hour during the polymerization, samples of the reaction medium are taken (0.1 ml diluted in 5 ml of methanol) in order to follow the progress of the reaction by steric exclusion chromatography.
- the reaction medium is diluted in one liter of water and then ultrafiltered through a membrane, the cut-off threshold of which is 100,000 Daltons.
- the polymer solution is then concentrated and then lyophilized.
- the yield is quite variable, generally around 60%.
- the macromolecules with an acrylamide skeleton are precipitated in acetone according to the following procedure:
- the reaction medium is slowly precipitated from 1 liter of acetone then filtered through a No. 4 frit and washed with three times 100 ml of acetone.
- the solid is recovered and then dried overnight with a vane pump.
- the mass yield is much better than for ultrafiltration-lyophilization and is close to 90%.
- the incorporation rate of the macromonomers was checked by NMR of hydrogen on the polymers diluted to 2g / 100ml in heavy water (Bruker apparatus at 250 and 400 MHz). It is found that the rate of incorporation depends, apart from the experimental fluctuations, only on the ratio between the initial mass of PNIPAM and the initial mass of hydrophilic monomer. It is, respectively, 6.5 +/- 0.3 mol%, for an initial concentration of 0.4 g of PNIPAM for 2.8 g of acrylamide, and 12 +/- 1 mol%, for a initial concentration of 0.8 g of PNIPAM per 2.8 g of acrylamide.
- each of the copolymers was introduced at a rate of 5 g / 100 ml in purified water (MilliQ).
- the viscosity of each of the corresponding solutions was measured on a cone-plan Brookfield DV3 rheometer controlled by Rheocalc software (Sodexim, Muizon, F).
- the retained shear rate is 10 (1 / s) for a temperature gradient of 1 ° C per minute. The results obtained are shown in Figure 1.
- the various synthesized copolymers indeed exhibit the rheological properties which make it possible to use them according to the invention, and in particular, that they have a viscosity V2 at a temperature T2 significantly higher, by a factor greater than 2 (100 %), often of order 10, and possibly up to a factor of 60, at the viscosity V1 obtained at a temperature T1 lower than T2 by at least 20 °.
- the temperature T1 may be between 20 and 40 ° C, or even lower, and the temperature T2 may be greater than 45 °, and preferably of the order of 60 ° C.
- the polymer T10AA prepared as a control according to the same protocol but without the PNIPAM macromonomer, and therefore being unable to exhibit in its structure the multiplicity of LCST blocks which characterizes the copolymers according to the invention, has a decreasing viscosity weakly and continuously as a function of the temperature, and therefore cannot exert the beneficial effects of the invention. Furthermore, for all of the copolymers and at a temperature change rate of 1 ° C. per minute, no significant hysteresis is observed, the viscosity curve being essentially identical as the temperature rises and falls.
- T10 in an ionic electrolyte such as for example potassium carbonate, or a buffer of the TRIS-TAPS 50 mM / Urea 7M type as used for sequencing.
- FIG. 2b are shown the behaviors of two solutions based on the PAM-NIPAM T10 copolymer, one for a concentration of 3 g / 100 ml of copolymer and the other for a concentration of 2 g / 100 ml of copolymer.
- the thermo-thickening character is sensitive from 2 g / 100ml, but reinforced to 3 g / 100ml and even more to 5 g / 100ml (figure 1), the concentration being also a parameter that will usefully be varied to adapt the media. according to the invention according to the particular applications.
- thermo-viscosifying character for concentrations less than or equal to 5 g / 100 ml, that is to say that they do not exhibit hysteresis. significant of their viscosity in temperature rise and fall, and are capable of flow in less than 30 s when the container is inverted.
- the T16 polymer gives rise to a gel-like state at 60 ° C. for concentrations of 8 g / 100 ml and more.
- separation media for capillary electrophoresis comprising, as copolymer, one of the copolymers prepared according to Example 2.
- the electrophoresis experiments presented in this and the following examples were performed using a laboratory-built apparatus, similar to that described in Lindberg et al., Electrophoresis, 18, 1973 (1997).
- the separated DNAs are detected by fluorescence with excitation by an Argon laser at 488 nm and emission at 530 +/- 30 nm.
- the injection is of the electrokinetic type.
- the capillary made of fused silica covered with polyimide (polymicro), with an internal diameter of 100 ⁇ m, is thermostatically controlled between the injection point and the detection point by circulation of silicone oil in a sealed envelope, with the exception of the first 2 and the last 2 centimeters. (unless otherwise stated, this type of capillary will be used in the electrophoresis experiments presented below)
- a medium comprising, as a copolymer, the PAM-NIPAM copolymer (T15) at a concentration of 2 g / 100 ml
- the medium is mixed with the TRIS-TAPS buffer (50 mM) and with the DNA marker (for SYBR GREEN I 10 "4 ).
- the capillary is filled at 25 ° C, the injection is carried out in 10 seconds at 25 volts per centimeter and the sample to be separated is of the same nature as that of the previous test.
- the separating properties of said medium are evaluated at two temperatures 25 ° C and 60 ° C and Figures 3a and 3b show these properties. It is found that the separation is both more resolving and faster at 60 ° C.
- the medium is introduced into the capillary at 25 ° C. at a concentration of 8 g / 100 ml mixed with the TRIS TAPS buffer (50 mM) and the DNA marker SYBR GREEN I (Molecular probes) diluted at 10 "4 relative to to the stock solution sold by the supplier.
- TRIS TAPS buffer 50 mM
- SYBR GREEN I Molecular probes
- a medium comprising, as copolymer, the PDMAM-NIPAM copolymer (T7) at a concentration of 2 g / 100 ml.
- This medium is mixed with the TRIS-acetate buffer (50 mM) and with the 10 "4 SYBR GREEN I DNA marker.
- the sample is the marker "Phi-X 174-
- Test 4-4 Separating properties of a medium comprising, as copolymer, the PAM-NIPAM T21 copolymer, prepared from the macromonomer PNIPAM-20, at a concentration of 2 g / 100 ml.
- the medium is mixed with the TRIS-acetate buffer (50 mM).
- the viscosity at high temperature is relatively low, and does not allow total suppression of the electroosmosis. Consequently, the capillary was prior to its use, washed with a 1 M hydrochloric acid solution comprising 1 g / 100 ml of polyvinylpirolidone of molecular mass 1,000,000 (Polysciences, Eppelheim, D).
- the sample is the "100 bp fluorescein ladder" marker, Bio-Rad, the fragments of which have sizes between 100 and 1000 bp, the injection is carried out in 10 seconds at 25 volts per centimeter.
- thermo-thickening character as it appears in the viscosity curves as a function of the temperature, is a characteristic advantage of the media according to the invention, and that the said viscosity curves can be used as a guide for optimization. separation properties.
- a separation medium comprising the PAM-NIPAM copolymer (T10).
- the copolymer is used at two different concentrations, 3 g / 100ml and 5 g / 100ml, respectively, in TRIS TAPS buffer (50 mM), 7M urea.
- the pH of the medium is of the order of 8.2.
- a test is carried out with a commercial sequencing medium (POP6 Perkin-Elmer), used as received.
- the capillary used has a length of 40 cm with an effective length of 30 cm and an internal diameter of 100 ⁇ m.
- the capillary is prior to its use, washed with a 1 M hydrochloric acid solution and comprising 1 g / 100 ml of polyvinyllpirolidone.de molecular weight 1,000,000.
- the separation medium is introduced into the capillary at 25 ° C.
- test sample is the product of a DNA sequence reaction ssM13mp18, ("T-terminated fragments"), prepared by cyclic sequencing ("cycle sequencing") with the fluorescein-primer kit distributed by Amersham, as indicated. supplied by the manufacturer.
- FIG. 6 show respectively the separating capacities of the commercial sequencing medium (control figure a), and media according to the invention based on the T10 copolymer at respective concentrations of 3 g / 100ml ( Figures b) and 5 g / 100ml (figures c) (the numbers above the peaks represent the length of the DNA fragment minus 48 bases)
- the media tested are based on either the PAM-NIPAM copolymer (T12) at a concentration of 8 g / 100ml, the PAM-NIPAM copolymer (T13) with a concentration of 5 g / 100ml or the DMAM-NIPAM copolymer (T7) with a concentration at 5 g / 100ml, respectively.
- Each of these media is of course supplemented with TRIS TAPS buffer (50 mM) and 7M urea. They have a pH of 8.2, the nature of the sample and the separation conditions are the same as those used in the previous test.
- the copolymer used at a concentration of 2 g / 100 ml is mixed with the TRIS-TAPS buffer (50 mM), 2 mM EDTA and the DNA marker
- the length of the capillary is 15 cm, including 10 cm to the detector, and the capillary is of the "DB17" (JW scientific) type with an internal diameter of 100 micrometers, in order to suppress the residual electroosmosis appearing with this medium.
- the sample is the High molecular weight standard marker (Life Technologies), and has fragments between 8271 and 48502 base pairs, the injection is carried out in 5 seconds at 100 volts per centimeter.
- the separation is carried out in pulsed fields with square pulses of +/- 200V, an asymmetry between the pulses + and - of 20% and a frequency of 30 Hz.
- the separation of the fragments can be carried out in less than 20 min ( Figure 8), against several hours in an ordinary entangled polymer solution (Heller et al. Electrophoresis, 16, 1423-1428 (1995).
- Separation properties and resolution at 40 ° C. of separation media in accordance with the invention contain polymers based on NIPAM macromonomers of different length (T22, T26, T24 and T25, respectively), dissolved at 3 g / 100ml in TRIS TAPS buffer (50 mM), 7M urea.
- the pH of the medium is of the order of 8.2.
- the sample used is a "50 BP LADDER- fluorescein Pharmacia.
- Test 4-9 Separation of sequence reaction products using a separation medium according to the invention at constant temperature.
- the medium tested is of the T16 type, at 5 g / 100 ml, in 50 mM Na TAPS buffer, 2 mM EDTA, 7M urea, the said medium being introduced into the capillary at 50 ° C, and the separation also taking place at 50 ° vs.
- the reading is carried out very well up to more than 500 bases, and that the introduction at low temperature is not in this precise case essential for the implementation of the medium according to the invention.
- This advantageous property comes from the fact that the medium is of the thermo-viscous type, and therefore does not present at the temperature at which the separation takes place, a gel type state which would prevent its introduction into the capillary.
- the polyvinyl alcohol constituting the water-soluble backbone of the copolymer is obtained beforehand by hydrolysis of polyvinyl acetate.
- the polymer used for the study has an acetate level of 12.4% by mole and a molar mass by weight of 145,000 g / mole. Its intrinsic viscosity in water at 30 ° C is 92 ml / g, the critical concentration for covering the C * chains is approximately 1.25 g / 100ml.
- the synthetic route followed is of the "grafting from" type. It is described by Nonaka & al. in a homogeneous medium (Y. Nonaka, Y. Ogata, S. Kurihara, Journal of Applied Polymer Science, vol.
- Table 3 below shows the characteristics of PVA-NIPAM obtained according to this protocol.
- This copolymer is prepared according to a protocol derived from that described by J.P. Kaczmarski and J.E. Glass, Langmuir, 1994, 10, 3035-3042.
- polyethylene glycol with a molecular weight of 35,000 (Merck, Hohenbrunn, D) (PEG) and 100 ml of anhydrous toluene are mixed under an argon atmosphere (low molecular weight polyoxyethylenes are commonly called “polyethylene glycol").
- PEG polyethylene glycol
- the mixture is heated to reflux under argon and approximately 10 to 15 ml of anhydrous toluene are evaporated.
- the solution is cooled to room temperature and 120 mg (0.2% by weight) of dibutyltin dilaurate are added to the solution.
- a stoichiometric amount of isophorone diisocyanate (128 mg) is dissolved in 5 ml of anhydrous toluene and added to the mixture.
- the reaction is followed by spectroscopy visualization
- the rheometer used in this example is identical to that used in Example 3 above.
- the PVA-NIPAM used corresponds to that of Table 3 and the linear triblock copolymer POP-POE-POP is that prepared in Example 6.
- Figure 9 are shown the rheological behaviors of media comprising
- the fluorescence detection is carried out under the same conditions as those described in Example 4.
- the capillary channel it has a total length of 40 cm and an effective length of 30 cm, an internal diameter of 100 ⁇ m and it is coated with an acrylamide derivative according to the method described in by HJERTEN J. Chromatogr., 1985, 347, 191 in order to suppress electroosmosis.
- the electrolyte comprises 4.75 g / 100 ml of the PVA-PNIPAM copolymer and 50 nM of TRIS TAPS buffer.
- the electric field is 200 volts per centimeter.
- the injection is carried out in 10 seconds at 200 volts per centimeter.
- the sample is the sizer 50-500, Pharmacia Biotech, diluted to 1/500 in MILI Q water (Miliport).
- the medium was tested under the conditions recommended for the medium described in Example 7 above.
- FIG. 11 a shows the separating properties of the control medium, that is to say simply based on unmodified polyethylene glycol, with
- FIG. 1 1b shows the separating properties of a medium comprising the POP-POE-POP copolymer at a temperature of 20 ° C by analogy to the control medium and
- FIG. 11c illustrates the separating properties of a medium based on said copolymer but at a temperature of 50 ° C.
- the copolymer exhibits from room temperature a resolution higher than that obtained with POE alone, undoubtedly because of its higher molecular weight, but that the properties are still greatly improved when we pass at 50 ° C., and shows that polymers of the block type having at least two blocks with LCST can also be advantageously used in the context of the invention for carrying out electrokinetic separations.
- PAAgNIPAM charged copolymer Polyacrylic Acid / Poly-N-lsopropylacrylamide
- the polyacrylic acid used is in solution at 12.5 g / 100ml in water.
- the polymer noted PAA500 has a weight-average molar mass of 500,000 g / mol (Fluka)
- the skeleton of the PAA500 is sensitive to pH; the ionizable carboxylic functions have a pKo around 4.25 pH units.
- the synthetic route followed consists in grafting short chains of NIPAM onto a weak fraction of the carboxylic acid functions of a polyacrylic acid skeleton. It breaks down into two stages:
- oligoNIPAM short chain of PNIPAM (Mw ⁇ 2000 g / mol) terminated by an amino function, and this by radical polymerization of NIPAM monomers in methanol in the presence of the transfer agent AET.HCI (2-aminoethanethiol hydrochlorinated) and the initiator AIBN (2,2'-azobistutyronitril) at 60 ° C for 20 hours, (this method of synthesis constitutes an alternative to that, in aqueous solution, used to prepare the polymers PNIPAM-A in the example 1).
- the oligoNIPAM of known length is then grafted onto the PAA500 skeleton reaction between acid and amino functional groups in the presence of a podiimide initiator:
- Table 4 shows the composition of the PAAgNIPAM copolymer obtained by this protocol.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Electrochemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Dispersion Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Compositions Of Macromolecular Compounds (AREA)
- Cell Separators (AREA)
- Graft Or Block Polymers (AREA)
- Electrostatic Separation (AREA)
- Other Resins Obtained By Reactions Not Involving Carbon-To-Carbon Unsaturated Bonds (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/869,382 US6830670B1 (en) | 1998-12-30 | 1999-12-28 | Heat-sensitive medium for separating species in a separating channel and use thereof |
EP99964728A EP1141693A1 (fr) | 1998-12-30 | 1999-12-28 | Milieu thermosensible pour la separation d'especes au sein d'un canal de separation |
CA002358092A CA2358092C (fr) | 1998-12-30 | 1999-12-28 | Milieu thermosensible pour la separation d'especes au sein d'un canal de separation |
AU30488/00A AU3048800A (en) | 1998-12-30 | 1999-12-28 | Heat-sensitive medium for separating species in a separator channel |
JP2000592627A JP2002534679A (ja) | 1998-12-30 | 1999-12-28 | 分離チャネルにおいて種を分離するための感熱性媒体 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR9816676A FR2788008B1 (fr) | 1998-12-30 | 1998-12-30 | Milieu thermosensible pour la separation electrocinetique d'especes au sein d'un canal de separation |
FR98/16676 | 1998-12-30 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2000040958A1 true WO2000040958A1 (fr) | 2000-07-13 |
Family
ID=9534715
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR1999/003304 WO2000040958A1 (fr) | 1998-12-30 | 1999-12-28 | Milieu thermosensible pour la separation d'especes au sein d'un canal de separation |
Country Status (7)
Country | Link |
---|---|
US (1) | US6830670B1 (fr) |
EP (1) | EP1141693A1 (fr) |
JP (1) | JP2002534679A (fr) |
AU (1) | AU3048800A (fr) |
CA (1) | CA2358092C (fr) |
FR (1) | FR2788008B1 (fr) |
WO (1) | WO2000040958A1 (fr) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2886301A1 (fr) * | 2005-05-25 | 2006-12-01 | Inst Curie | Nouveau procede de preparation de copolymeres en peigne hydrosolubles |
US7932339B2 (en) | 2005-05-25 | 2011-04-26 | Centre National De La Recherche Scientifique | Method for producing water-soluble comb-shaped copolymers |
WO2013186455A1 (fr) | 2012-06-15 | 2013-12-19 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Nouveau copolymère en peigne et procédé pour sa préparation |
WO2014096595A1 (fr) | 2012-12-21 | 2014-06-26 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Nouveaux polymères ampholytes à caractère thermosensible |
WO2014096594A1 (fr) | 2012-12-21 | 2014-06-26 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Nouveau procédé de préparation de polymères thermo-épaississants et nouveaux copolymères en peigne |
Families Citing this family (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2810905B1 (fr) * | 2000-06-30 | 2003-05-16 | Inst Curie | Additif pour minimiser les phenomenes d'adsorption et/ou d'electroosmose |
US8975328B2 (en) | 2000-06-30 | 2015-03-10 | Institute Curie | Non-thermosensitive medium for analyzing species in a channel and for minimizing adsorption and/or electroosomosic phenomena |
FR2811083B1 (fr) | 2000-06-30 | 2002-11-22 | Inst Curie | Milieu liquide non-thermosensible pour l'analyse d'especes au sein d'un canal |
FR2826660B1 (fr) * | 2001-06-27 | 2003-08-15 | Seppic Sa | Nouveaux polymeres, le procede pour leur preparation, microlatex inverses et latex inverses les contenant et leur utilisation comme thermoepaississant |
JP4677524B2 (ja) * | 2004-12-13 | 2011-04-27 | 独立行政法人産業技術総合研究所 | 微細管電気泳動による微生物分離用分離促進剤及び分析装置 |
NO20053226D0 (no) * | 2005-06-30 | 2005-06-30 | Uni I Oslo | Fremgangsmate for elektrokinetisk migrasjon. |
KR20080036217A (ko) * | 2005-08-26 | 2008-04-25 | 신세스 게엠바하 | 수핵용 수화겔 풍선 보철물 |
FR2902799B1 (fr) * | 2006-06-27 | 2012-10-26 | Millipore Corp | Procede et unite de preparation d'un echantillon pour l'analyse microbiologique d'un liquide |
US8163886B2 (en) * | 2006-12-21 | 2012-04-24 | Emd Millipore Corporation | Purification of proteins |
US8362217B2 (en) | 2006-12-21 | 2013-01-29 | Emd Millipore Corporation | Purification of proteins |
US8569464B2 (en) | 2006-12-21 | 2013-10-29 | Emd Millipore Corporation | Purification of proteins |
WO2009020682A2 (fr) | 2007-05-08 | 2009-02-12 | The Trustees Of Boston University | Fonctionnalisation chimique d'ensembles de nanopores et de nanopores à semi-conducteurs, et leurs applications |
FR2932070B1 (fr) | 2008-06-10 | 2012-08-17 | Oreal | Ensemble de maquillage et/ou de soin des cils |
US8999702B2 (en) | 2008-06-11 | 2015-04-07 | Emd Millipore Corporation | Stirred tank bioreactor |
US20100190963A1 (en) * | 2008-12-16 | 2010-07-29 | Millipore Corporation | Stirred Tank Reactor And Method |
FR2940761B1 (fr) | 2009-01-07 | 2012-12-28 | Polymerexpert Sa | Composition anti-ronflement contenant un polymere thermogelifiant |
WO2011040996A1 (fr) | 2009-09-30 | 2011-04-07 | Quantapore, Inc. | Séquençage ultrarapide de polymères biologiques au moyen de nanopores marqués |
ES2754210T3 (es) | 2010-05-17 | 2020-04-16 | Emd Millipore Corp | Polímeros sensibles a estímulos para la purificación de biomoléculas |
US9408785B2 (en) | 2012-10-15 | 2016-08-09 | L'oreal | Hair styling compositions containing aqueous wax dispersions |
US10626294B2 (en) | 2012-10-15 | 2020-04-21 | L'oreal | Aqueous wax dispersions containing volatile solvents |
US10413496B2 (en) | 2012-10-15 | 2019-09-17 | L'oreal | Aqueous wax dispersions |
US9651539B2 (en) | 2012-10-28 | 2017-05-16 | Quantapore, Inc. | Reducing background fluorescence in MEMS materials by low energy ion beam treatment |
EP3004385B1 (fr) | 2013-05-24 | 2018-11-28 | Quantapore Inc. | Analyse d'acides nucléiques basés sur des nanopores avec une détection par fret mixte |
US10561596B2 (en) | 2014-04-11 | 2020-02-18 | L'oreal | Compositions and dispersions containing particles comprising a polymer |
CA2963604C (fr) | 2014-10-10 | 2023-02-14 | Quantapore, Inc. | Analyse de polymeres, a base de nanopore, a l'aide de marqueurs fluorescents a desactivation mutuelle |
CN107002126B (zh) | 2014-10-24 | 2021-05-25 | 昆塔波尔公司 | 使用纳米结构阵列的聚合物的高效光学分析 |
JP2019522983A (ja) | 2016-07-05 | 2019-08-22 | クアンタポール, インコーポレイテッド | 光学ベースのナノポア配列決定 |
CN109225175B (zh) * | 2018-08-21 | 2021-07-20 | 江苏大学 | 一种磁性复合微球及其制备方法和应用 |
FR3091996B1 (fr) | 2019-01-24 | 2021-01-29 | Les Laboratoires Brothier | Composition cicatrisante |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0583814A1 (fr) * | 1992-08-20 | 1994-02-23 | Sofitech N.V. | Polymères thermoviscosifiants, leur synthèse et leurs applications notamment dans l'industrie pétrolière |
WO1995030782A1 (fr) * | 1994-05-10 | 1995-11-16 | Soane Biosciences | Supports de separation utilises dans l'electrophorese sur gel |
WO1997009400A1 (fr) * | 1995-09-08 | 1997-03-13 | Rhone-Poulenc Chimie | Utilisation en tant qu'agents thermoepaississants de copolymeres multiblocs |
WO1998010274A1 (fr) * | 1996-09-04 | 1998-03-12 | The Research Foundation Of State University Of New York | Nouveaux milieu de separation et traitement de surface destines a un appareillage d'electrophorese |
Family Cites Families (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH03296657A (ja) * | 1990-04-13 | 1991-12-27 | W R Grace & Co | 電気泳動用支持体およびそれを用いた電気泳動法 |
US5196099A (en) * | 1990-04-13 | 1993-03-23 | W. R. Grace & Co.-Conn. | Electrophoretic matrix and electrophoresis using same |
JPH04278451A (ja) * | 1991-02-27 | 1992-10-05 | W R Grace & Co | 分離回収用電気泳動ゲルおよびそれを用いた分離回収法 |
JPH04278452A (ja) * | 1991-02-27 | 1992-10-05 | W R Grace & Co | 分離回収用電気泳動ゲルおよびそれを用いた分離回収法 |
US5238545A (en) * | 1991-02-27 | 1993-08-24 | W. R. Grace & Co.-Conn. | Electrophoretic gel for separation and recovery of substances and its use |
JPH05133937A (ja) * | 1991-10-08 | 1993-05-28 | W R Grace & Co | 電気泳動用支持体およびそれを用いた電気泳動法 |
US5885432A (en) * | 1992-11-05 | 1999-03-23 | Soane Biosciences | Un-crosslinked polymeric media for electrophoresis |
JP3359370B2 (ja) * | 1993-03-17 | 2002-12-24 | 光弘 清水 | 分離回収用電気泳動ゲルおよびそれを用いた分離回収法 |
US5883211A (en) * | 1996-01-19 | 1999-03-16 | Aclara Biosciences, Inc. | Thermoreversible hydrogels comprising linear copolymers and their use in electrophoresis |
-
1998
- 1998-12-30 FR FR9816676A patent/FR2788008B1/fr not_active Expired - Fee Related
-
1999
- 1999-12-28 JP JP2000592627A patent/JP2002534679A/ja active Pending
- 1999-12-28 AU AU30488/00A patent/AU3048800A/en not_active Abandoned
- 1999-12-28 WO PCT/FR1999/003304 patent/WO2000040958A1/fr active Application Filing
- 1999-12-28 US US09/869,382 patent/US6830670B1/en not_active Expired - Fee Related
- 1999-12-28 CA CA002358092A patent/CA2358092C/fr not_active Expired - Fee Related
- 1999-12-28 EP EP99964728A patent/EP1141693A1/fr not_active Withdrawn
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0583814A1 (fr) * | 1992-08-20 | 1994-02-23 | Sofitech N.V. | Polymères thermoviscosifiants, leur synthèse et leurs applications notamment dans l'industrie pétrolière |
WO1995030782A1 (fr) * | 1994-05-10 | 1995-11-16 | Soane Biosciences | Supports de separation utilises dans l'electrophorese sur gel |
WO1997009400A1 (fr) * | 1995-09-08 | 1997-03-13 | Rhone-Poulenc Chimie | Utilisation en tant qu'agents thermoepaississants de copolymeres multiblocs |
WO1998010274A1 (fr) * | 1996-09-04 | 1998-03-12 | The Research Foundation Of State University Of New York | Nouveaux milieu de separation et traitement de surface destines a un appareillage d'electrophorese |
Non-Patent Citations (3)
Title |
---|
D. HOURDET: "THERMOTHICKENING POLYELECTROLYTES", POLYMER PREPRINTS, vol. 34, no. 1, 1993, pages 972 - 973, XP002100609 * |
HOURDET D ET AL: "Synthesis of thermoassociative copolymers", POLYMER, vol. 38, no. 10, May 1997 (1997-05-01), pages 2535-2547, XP004059755 * |
L'ALLORET F ET AL: "REVERSIBLE THERMOASSOCIATION OF WATER-SOLUBLE POLYMERS", REVUE DE L'INSTITUT FRANCAIS DU PETROLE, vol. 52, no. 2, March 1997 (1997-03-01), pages 117 - 128, XP000703457 * |
Cited By (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2886301A1 (fr) * | 2005-05-25 | 2006-12-01 | Inst Curie | Nouveau procede de preparation de copolymeres en peigne hydrosolubles |
WO2007000535A2 (fr) * | 2005-05-25 | 2007-01-04 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Nouveau procede de preparation de copolymeres en peigne hydrosolubles |
WO2007000535A3 (fr) * | 2005-05-25 | 2007-03-15 | Seppic Sa | Nouveau procede de preparation de copolymeres en peigne hydrosolubles |
US7932339B2 (en) | 2005-05-25 | 2011-04-26 | Centre National De La Recherche Scientifique | Method for producing water-soluble comb-shaped copolymers |
WO2013186455A1 (fr) | 2012-06-15 | 2013-12-19 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Nouveau copolymère en peigne et procédé pour sa préparation |
US9884933B2 (en) | 2012-06-15 | 2018-02-06 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Comb copolymer and process for the preparation thereof |
WO2014096595A1 (fr) | 2012-12-21 | 2014-06-26 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Nouveaux polymères ampholytes à caractère thermosensible |
WO2014096594A1 (fr) | 2012-12-21 | 2014-06-26 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Nouveau procédé de préparation de polymères thermo-épaississants et nouveaux copolymères en peigne |
FR3000077A1 (fr) * | 2012-12-21 | 2014-06-27 | Seppic Sa | Nouveau procede de preparation de polymeres thermo-epaississants et nouveaux copolymeres en peigne |
FR3000078A1 (fr) * | 2012-12-21 | 2014-06-27 | Seppic Sa | Nouveaux polymeres ampholytes a caractere thermosensible |
US10131729B2 (en) | 2012-12-21 | 2018-11-20 | Societe D'exploitation De Produits Pour Les Industries Chimiques Seppic | Ampholytic polymers having a thermosensitive character |
Also Published As
Publication number | Publication date |
---|---|
FR2788008B1 (fr) | 2001-03-23 |
EP1141693A1 (fr) | 2001-10-10 |
CA2358092A1 (fr) | 2000-07-13 |
CA2358092C (fr) | 2007-09-11 |
JP2002534679A (ja) | 2002-10-15 |
US6830670B1 (en) | 2004-12-14 |
AU3048800A (en) | 2000-07-24 |
FR2788008A1 (fr) | 2000-07-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2358092C (fr) | Milieu thermosensible pour la separation d'especes au sein d'un canal de separation | |
US7399396B2 (en) | Sparsely cross-linked nanogels: a novel polymer structure for microchannel DNA sequencing | |
US5290418A (en) | Viscous electrophoresis polymer medium and method | |
US5759369A (en) | Viscous electrophoresis polymer medium and method | |
Sudor et al. | New block‐copolymer thermoassociating matrices for DNA sequencing: Effect of molecular structure on rheology and resolution | |
US6926815B2 (en) | Copolymers for capillary gel electrophoresis | |
US20100187111A1 (en) | Graft Copolymers, Their Preparation And Use in Capillary Electrophoresis | |
EP1951413B1 (fr) | Systèmes de polymères matriciels et de polymères dynamiques, et compositions pour la séparation de microcanal | |
US20180024095A1 (en) | Non-thermosensitive medium for analyzing species in a channel and for minimizing adsorption and/or electroosomosic phenomena | |
Barbier et al. | Comb‐like copolymers as self‐coating, low‐viscosity and high‐resolution matrices for DNA sequencing | |
EP1360215A2 (fr) | Solution de traitement de surface minimisant les phenomenes d'adsorption et/ou d'electroosmose | |
Chiari et al. | Separation of DNA fragments in hydroxylated poly (dimethylacrylamide) copolymers | |
Chiari et al. | Separation of oligonucleotides and DNA fragments by capillary electrophoresis in dynamically and permanently coated capillaries, using a copolymer of acrylamide and β‐d‐glucopyranoside as a new low viscosity matrix with high sieving capacity | |
EP0156657A1 (fr) | Copolymères réticulés non ioniques, partiellement hydrophiles, et leur application à la chromatographie d'exclusion en milieu aqueux | |
US9347915B2 (en) | Non-thermosensitive medium for analyzing species in a channel and for minimizing absorption and/or electroosomosic phenomena | |
Chiari et al. | Vinylpyrrolidine‐β‐cyclodextrin copolymer: A novel chiral selector for capillary electrophoresis | |
Bromberg | Polyacrylamide conjugated with poly (oxyethylene)‐block‐poly (oxypropylene)‐block‐poly (oxyethylene): a self‐assembling material | |
Zhang et al. | Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis | |
US7037978B2 (en) | Quasi-interpenetrating networks used as separation media | |
FR2887787A1 (fr) | Utilisation de polymeres reactifs pour moduler et controler les flux electrocinetiques dans un dispositif micro-ou nanofluidique | |
CA2538505A1 (fr) | Reseaux quasi-interpenetrants utilises comme milieux de separation | |
Li et al. | DNA Separation by Microchip Electrophoresis Using Copolymers of Poly (vinylpyrrolidone) and Hydroxyethylcellulose |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZA ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1999964728 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2358092 Country of ref document: CA Ref country code: CA Ref document number: 2358092 Kind code of ref document: A Format of ref document f/p: F |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 2000 592627 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09869382 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 1999964728 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
CFP | Corrected version of a pamphlet front page |
Free format text: REVISED ABSTRACT RECEIVED BY THE INTERNATIONAL BUREAU AFTER COMPLETION OF THE TECHNICAL PREPARATIONS FOR INTERNATIONAL PUBLICATION |