U.S. patent application number 10/312538 was filed with the patent office on 2004-05-06 for non-thermosensitive medium for analysing species inside a channel.
Invention is credited to Barbier, Valessa, Viovy, Jean-Louis.
Application Number | 20040084310 10/312538 |
Document ID | / |
Family ID | 8851970 |
Filed Date | 2004-05-06 |
United States Patent
Application |
20040084310 |
Kind Code |
A1 |
Viovy, Jean-Louis ; et
al. |
May 6, 2004 |
Non-thermosensitive medium for analysing species inside a
channel
Abstract
The invention concerns a non-thermosensitive liquid medium for
analyzing, purifying or separating species in a channel and
comprising at least a polymer consisting of several polymeric
segments. The invention is characterised in that the polymer is of
the irregular block-copolymer or irregular comb-like polymer type
and has on the average at least three junction points between
polymeric segments of different chemical or topological nature. The
invention also concerns methods for analyzing, purifying or
separating species using a non-thermosenstive separation
medium.
Inventors: |
Viovy, Jean-Louis; (Paris,
FR) ; Barbier, Valessa; (Paris, FR) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET 2ND FLOOR
ARLINGTON
VA
22202
|
Family ID: |
8851970 |
Appl. No.: |
10/312538 |
Filed: |
July 14, 2003 |
PCT Filed: |
June 29, 2001 |
PCT NO: |
PCT/FR01/02103 |
Current U.S.
Class: |
204/450 |
Current CPC
Class: |
C08F 290/046 20130101;
C08F 2/10 20130101; C08F 2810/40 20130101; C08F 120/54 20130101;
C08F 290/04 20130101; C08F 220/56 20130101; C08F 8/10 20130101;
C08F 265/10 20130101; C08F 120/56 20130101; C08F 2810/30 20130101;
G01N 27/44747 20130101; C08F 290/046 20130101; C08F 8/10
20130101 |
Class at
Publication: |
204/450 |
International
Class: |
C02F 001/469; C25B
007/00; B01D 061/42 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 30, 2000 |
FR |
00/08526 |
Claims
1. A non-thermosensitive liquid medium for analyzing, purifying or
separating species inside a channel, and comprising at least one
polymer composed of several polymer segments, characterized in that
said polymer is of the irregular block copolymer type or irregular
comb polymer type and has on average at least three junction points
established between polymer segments of different chemical or
topological nature.
2. The medium as claimed in claim 1, characterized in that all the
segments of at least one type of chemical or topological nature
forming part of the composition of said polymer have a
polydispersity of at least 1.5.
3. The medium as claimed in claim 1 or 2, characterized in that the
segments of each of the types of chemical or topological nature
forming part of the composition of said polymer have a
polydispersity of at least 1.5.
4. The medium as claimed in claim 2 or 3, characterized in that
said polydispersity is greater than 1.8.
5. The medium as claimed in any one of the preceding claims,
characterized in that said polymer has an average molecular mass of
greater than 50 000 and preferably greater than 300 000.
6. The medium as claimed in any one of claims 1 to 5, characterized
in that said polymer shows specific affinity for the walls of said
channel.
7. The medium as claimed in claim 6, characterized in that said
polymer has at least one type of polymer segments showing, within
the separating medium, specific affinity for the wall, and at least
one type of polymer segment showing in said medium less or no
affinity for the wall.
8. The medium as claimed in claim 6 or 7, characterized in that all
the segments showing specific affinity for the wall represent
between 2% and 80% by mass of the average total molar mass of said
polymer.
9. The medium as claimed in one of the preceding claims,
characterized in that the polymer shows specific affinity for one
or more analytes.
10. The medium as claimed in claim 9, characterized in that the
polymer bears nucleotides or polypeptides of determined
sequence.
11. The medium as claimed in claim 9, characterized in that the
polymer is combined with a protein, a protein fraction, a protein
complex and/or an acidic or basic function.
12. The medium as claimed in one of the preceding claims,
characterized in that all the polymer segments of at least one type
of chemical or topological nature have on average a number of atoms
of greater than 75, and preferably greater than 210, or have a
molecular mass of greater than 1 500 and preferably greater than 4
500.
13. The medium as claimed in any one of the preceding claims,
characterized in that the various types of polymer segments of
which said polymer is composed have on average an average number of
atoms of greater than 75, and preferably greater than 210, or have
a molecular mass of greater than 1 500 and preferably greater than
4 500.
14. The medium as claimed in any one of the preceding claims,
characterized in that the polymer has on average a number of
junction points of between 4 and 100.
15. The medium as claimed in any one of the preceding claims,
characterized in that the polymer is a block copolymer containing
on average at least four polymer segments.
16. The medium as claimed in any one of the preceding claims,
characterized in that the polymer is a comb polymer containing on
average at least two side chains.
17. The medium as claimed in any one of the preceding claims,
characterized in that the polymer comprises at least one type of
segment chosen from polyethers, polyesters, for instance
polyglycolic acid, soluble random homopolymers and copolymers of
the polyoxyalkylene type, for instance polyoxypropylene,
polyoxybutylene or polyoxyethylene, polysaccharides, polyvinyl
alcohol, polyvinylpyrrolidone, polyurethanes, polyamides,
polysulfonamides, polysulfoxides, polyoxazoline, polystyrene
sulfonate, and substituted or unsubstituted acrylamide,
methacrylamide and allyl polymers and copolymers.
18. The medium as claimed in any one of the preceding claims,
characterized in that said polymer comprises at least one polymer
chosen from: copolymers of the comb copolymer type, the skeleton of
which is of dextran, acrylamide, acrylic acid, acryloylaminoethanol
or (N,N)-dimethylacrylamide type and onto which are grafted side
segments of acrylamide, substituted acrylamide or
(N,N)-dimethylacrylamide (DMA) type, or of the DMA/allyl glycidyl
ether (AGE) copolymer type, or of homopolymer or copolymer of
oxazoline or of oxazoline derivatives; non-thermosensitive
copolymers of the irregular sequential copolymer type having along
their skeleton an alternation of segments of polyoxyethylene type
and of segments of polyoxypropylene type, or an alternation of
segments of polyoxyethylene type and of segments of polyoxybutylene
type, or an alternation of segments of polyethylene and of segments
of polyether type that are more hydrophobic than polyoxyethylene;
copolymers of the irregular sequential block copolymer type having
along their skeleton an alternation of segments of acrylamide,
acrylic acid, acryloylaminoethanol or dimethylacrylamide type, on
the one hand, and segments of (N,N)-dimethylacrylamide (DMA) type,
or of DMA/allyl glycidyl ether (AGE) copolymer type, or of
homopolymer or copolymer of oxazoline or of oxazoline derivatives;
polymers of the irregular comb polymer type, the skeleton of which
is of the agarose, acrylamide, substituted acrylamide, acrylic
acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl
glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer
type, of oxazoline and oxazoline derivative, of dextran, of
methylcellulose, of hydroxyethylcellulose, of modified cellulose,
of polysaccharide or of ether oxide type, and onto which are
grafted side segments of agarose, acrylamide, substituted
acrylamide, acrylic acid, acryloylaminoethanol, dimethylacrylamide
(DMA), or allyl glycidyl ether (AGE) polymer type, of DMA/AGE
random copolymer type, of oxazoline and oxazoline derivative, of
dextran, of methylcellulose, of hydroxyethylcellulose, of modified
cellulose, of polysaccharide or of ether oxide type; copolymers of
the irregular comb copolymer type, the skeleton of which is of the
acrylamide, substituted acrylamide, acrylic acid,
acryloylaminoethanol, dimethylacrylamide (DMA), or allyl glycidyl
ether (AGE) polymer type, of DMA/AGE random copolymer type, of
oxazoline and oxazoline derivative, of dextran, of agarose, of
methylcellulose, of hydroxyethylcellulose, of modified cellulose,
of polysaccharide or of ether oxide type, and bears short-chain
hydrophobic side segments such as alkyl chains, aromatic
derivatives, fluoroalkyls, silanes or fluorosilanes.
19. The use of a medium as claimed in one of claims 1 to 18, for
separating, purifying, filtering or analyzing species chosen from
molecular or macromolecular species, biological macromolecules of
nucleic acid type, their synthesis analogs, proteins, polypeptides,
glycopeptides and polysaccharides, organic molecules, synthetic
macromolecules or particles such as mineral particles, lattices,
cells or organelles.
20. The use as claimed in claim 19 or the use of a medium as
claimed in one of claims 1 to 18, in a channel of which at least
one dimension is submillimetric.
21. The use as claimed in claim 19 or 20 or the use of a medium as
claimed in one of claims 1 to 18, for electrokinetic
separations.
22. The use as claimed in one of claims 19 to 21 or the use of a
medium as claimed in one of claims 1 to 18, for diagnosis, gene
typing, and high-throughput screening, quality control, or for
detecting the presence of genetically modified organisms in a
product.
23. A process for separating, analyzing and/or identifying species
contained in a sample, characterized in that it comprises a/ the
filling of the channel of a separating device with a separating
medium as claimed in one of claims 1 to 18, b/ the introduction of
said sample containing said species into one end of said channel,
c/ the application of an external field intended to move certain
species contained in the sample, and d/ the recovery of said
species or the detection of their passage at a point along the
channel that is different from the point of introduction of the
sample.
Description
[0001] The present invention relates to the field of techniques for
analyzing, separating and purifying species, according to which it
is necessary to migrate these species in a fluid known as the
"separating medium".
[0002] The invention is directed more particularly toward proposing
a separating medium that is suitable for separating species in
channels or capillaries, at least one of the dimensions of which is
submillimetric, and typically between 20 .mu.m and 200 .mu.m
(referred to hereinbelow as microchannels). The invention in
particular concerns methods for separating or analyzing biological
macromolecules by capillary electrophoresis, by chromatography or
by any method used in microchannels (capillary electrophoresis and
capillary chromatography, microfluid systems, and "chip
laboratories"). The invention is particularly useful in the case of
electrophoresis.
[0003] In the text hereinbelow, the expression "microfluid system"
will denote any system in which fluids and/or species contained in
a fluid are moved inside a channel or a set of channels, at least
one of the dimensions of which is submillimetric, and the term
"capillary electrophoresis (CE)" will denote microfluid systems in
which the transportation of species is performed by the action of
an electric field.
[0004] CE and microfluid systems allow faster separations with
higher resolutions than the older methods of gel electrophoresis,
do not require an anticonvective medium, and their properties have
been used widely to perform separations of ions in liquid medium.
At the present time, the vast majority of separations of biological
macromolecules performed by CE use solutions of interlocked linear
water-soluble polymers that have the advantage of being able to be
replaced as often as necessary.
[0005] Many noncrosslinked polymers have been proposed as media for
separating species inside a channel, in particular in the context
of capillary electrophoresis. The choice of the best polymer for a
given application depends on several parameters. For example, for
the separation of analytes as a function of their sizes, it is
necessary for the medium to present the analytes with sufficiently
resistant topological obstacles (Viovy et al., Electrophoresis,
1993, 14, 322). This involves the separating medium being highly
interlocked, and thus relatively viscous. It is also necessary for
the polymers present in the separating medium not to undergo any
interactions of attraction with the analytes. The reason for this
is that interactions of this type give rise to a slowing-down of
certain analytes, and to additional dispersion (H. Zhou et al. HPCE
2000, Saarbrucken, 20-24/2/2000). Thus, it is well known that for
DNA sequencing, or for protein separation, poorer results are
obtained when the matrix has a more hydrophobic nature.
[0006] It has also been proposed in the literature to use
copolymers as separating medium. In Menchen, WO 94/07133, it is
proposed to use as separating medium in capillary electrophoresis,
media comprising copolymers of block copolymer type which are said
to be "regular" since they have hydrophilic segments of a selected
and essentially uniform length and a plurality of regularly spaced
hydrophobic segments, at a concentration higher than the overlap
concentration between polymers. These media have the advantage of
being shear-thinning, i.e. they can be introduced into a capillary
under high pressure, while at the same time presenting solid
topological obstacles in the absence of external pressure.
Unfortunately, the media that may be used according to this
principle are difficult to synthesize, which makes them expensive
and limits the type of structures that may be envisaged. Also,
these polymers are relatively hydrophobic, and their performance
qualities for DNA sequencing, for example, are mediocre.
[0007] It has also been proposed to use as separating media
thermosensitive media, the viscosity of which varies greatly during
an increase in temperature. This type of medium has the advantage
of allowing the injection of said medium into the capillary at a
first temperature in a state of low viscosity, and the separation
at a second temperature in a state of higher viscosity that
displays good separation efficiency, as is commonly performed in
gel electrophoresis, in particular with agarose. Patent
applications WO 94/10561 and WO 95/30782 especially propose media
that allow an easier injection by raising the temperature. In point
of fact, said patent applications essentially describe microgels
capable of decreasing in volume at high temperature (thus leading
to a dilute solution of discontinuous particles of low viscosity)
and of swelling at low temperature until they entirely fill the
separating channel (thus giving the medium a gelled nature and good
separating properties). Patent application WO 98/10274 itself
proposes a molecular separating medium comprising at least one type
of block copolymers that is in solution at a first temperature and
in a gel-type state at a second temperature. The media described
comprise triblock polymers of low molecular masses (typically less
than 20 000), of the
polyoxyethylene-polyoxypropylene-polyoxyethylene (POE-POP-POE)
family and more specifically (POE.sub.99-POP.sub.69-POE.sub.99 in
which the indices represent the number of monomers of each block)
(trade name "Pluronic F127"). At low temperature, the two POE
segments at the ends of the triblock systems are water-soluble and,
given the low molecular mass of the copolymer, the solutions are
relatively nonviscous up to a high concentration. By raising the
temperature by about 15-25.degree. C., the central POP segment
becomes more hydrophobic, and these polymers become associated to
form a gel-type state. However, this mechanism presents several
drawbacks in electrophoresis. Firstly, it gives rise to a gel state
that has good electrophoretic separating properties only at high
polymer concentrations, of greater than 15 g/100 ml or even 20
g/100 ml, which leads to high friction and long migration times.
Moreover, the dependence of the properties as a function of the
rate of change of temperature makes the reproducibility of the
results random. Finally, for many applications and in many devices,
it is inconvenient, or even impossible, to change the temperature
between the stage of filling of the channel and the separating
stage.
[0008] In Madabhushi, U.S. Pat. No. 5,552,028, WO 95/16910 and WO
95/16911, it is also proposed to use separating media comprising a
screening medium and a surface-interaction component consisting of
a polymer with wall-adsorption properties, with a molecular mass of
between 5 000 and 1 000 000, of the disubstituted acrylamide
polymer type. These matrices, and more particularly
polydimethylacrylamide (PDMA), make it possible to reduce the
electroosmosis and in certain applications, for instance
sequencing, lead to good separating properties. However, they are
relatively hydrophobic, which limits their performance qualities
for certain applications, for instance DNA sequencing, and is even
more harmful for other applications, for instance protein
separation. Moreover, they lead to slow separations.
[0009] Consequently, despite the large number of studies and
systems proposed, a medium that is optimum for all the various
aspects of cost, of separation efficiency, of reduction of
interactions with the walls and of convenience of use is not
available at the present time for all the applications mentioned
above.
[0010] One object of the present invention is, precisely, to
propose the use of a family of polymers that is particularly
advantageous as non-thermosensitive liquid separating medium for
the separation, analysis or purification of species in
channels.
[0011] More particularly, a subject of the present invention is a
non-thermosensitive liquid medium for analyzing, purifying or
separating species inside a channel, and comprising at least one
polymer composed of several polymer segments, characterized in that
said polymer is of the irregular block copolymer type or irregular
comb polymer type and has on average at least three junction points
established between polymer segments of different chemical or
topological nature.
[0012] For the purposes of the invention, the term "polymer"
denotes a product consisting of a set of macromolecules and
characterized by certain properties such as molecular mass,
polydispersity, chemical composition and microstructure. The
polydispersity characterizes the molecular mass distribution of the
macromolecules, in the meaning of the mass-average familiar to
those skilled in the art. The term "microstructure" means the way
in which the monomers forming part of the chemical composition of
the macromolecules are arranged within the latter.
[0013] According to the invention, the term "liquid" means, as
opposed to a "gel", any condensed medium capable of flowing,
whether it is newtonian or viscoelastic.
[0014] In the present case, gels derived from the copolymerization
of monomers in the presence of difunctional or multifunctional
crosslinking agent(s) are excluded from the field of the invention.
The reason for this is that, given their crosslinked state, these
gels are solid or elastic and are therefore not liquid. In
particular, they are unsuitable for introduction into a
capillary.
[0015] The liquid medium according to the invention is
non-thermosensitive, i.e. it does not display, between its
solidification point plus 10.degree. C., and its boiling point
minus 10.degree. C., a sudden change in its viscosity. The term
"sudden change" means a variation by a factor of 2 or more, over a
temperature range of 20.degree. C. or less.
[0016] For the purposes of the invention, the expression
"separation method" is intended to cover any method directed toward
separating, purifying, identifying or analyzing all or some of the
species contained in a sample. In this case, the liquid is referred
to as the "separating medium" and through it pass the species to be
separated or at least some of them in the course of the separation
process.
[0017] The term "species" is generally intended to denote
particles, organelles or cells, molecules or macromolecules, and in
particular biological macromolecules, for instance nucleic acids
(DNA, RNA or oligonucleotides), nucleic acid analogs obtained by
synthesis or chemical modification, proteins, polypeptides,
glycopeptides and polysaccharides. In analytical methods, said
species are commonly referred to as "analytes".
[0018] The invention is particularly advantageous in the case of
electrokinetic separation methods.
[0019] The term "electrokinetic separation" is intended to cover
any method directed toward separating all or some of the species
contained in a mixture by making them migrate in a medium by the
action of an electric field, whether the field exerts its motor
action on the analytes directly or indirectly, for example by means
of a displacement of the medium itself, for instance in
electrochromatography, or by means of a displacement of associated
species such as micells, in the case of micellar
electrochromatography, or by any combination of direct and indirect
actions. Any separation method in which said action of the electric
field is combined with another motor action of nonelectric origin
are also considered as an electrokinetic separation method
according to the invention. Accordingly, methods of capillary
electrophoresis or of electrophoresis on "chips" are referred to as
"electrokinetic".
[0020] Advantageously, in particular in the case of electrokinetic
separations, the liquid will consist of an electrolyte.
[0021] For the purposes of the invention, the term "electrolyte"
denotes a liquid capable of conducting ions. In the most common
case, this medium is a buffered aqueous medium, for instance
buffers based on phosphate, tris(hydroxymethyl)aminomethane (TRIS),
borate, N-tris(hydroxymethyl)meth- yl-3-aminopropanesulfonic acid
(TAPS), histidine, lysine, etc. Numerous examples of buffers that
may be used in electrophoresis are known to those skilled in the
art, and a certain number of them are described, for example, in
Sambrook et al., "Molecular Cloning: a laboratory manual", Cold
Spring Harbor Lab, New York, 1989. However, any type of electrolyte
may be used in the context of the invention, especially
aqueous-organic solvents such as, for example, water-acetonitrile,
water-formamide or water-urea mixtures, or polar organic solvents
such as, also by way of example, N-methylformamide. The "sequencing
buffer" electrolytes consisting of an aqueous buffer at alkaline pH
containing an appreciable proportion of urea and/or of formamide
are found to be particularly useful in the context of the
invention.
[0022] The term "channel" denotes any volume delimited by one or
more solid walls, having at least two orifices and intended to
contain a fluid or to have a fluid pass through it.
[0023] The invention is particularly advantageous in systems
comprising at least one channel, at least one dimension of which is
submillimetric, such as capillary electrokinetic separation
systems, microfluid systems and, more generally, systems for
separating species using microchannels.
[0024] According to one preferred variant, the invention is
directed toward the use, as a liquid separating medium, of a
solution containing polymers having on average at least four
junction points, preferably a number of junction points of between
4 and 100 and more preferably a number of junction points of
between 4 and 40.
[0025] The term "junction point" means a point connecting either
two polymer segments of significantly different chemical nature,
for instance in the case of a block copolymer, or a point of
crosslinking between more than two polymer segments of identical or
different chemical nature, for instance in comb polymers. By way of
example, a comb polymer bearing three side branches comprises three
junction points and seven separate polymer segments. Similarly, a
sequential block copolymer of A-B-A-B type comprises three junction
points and four separate polymer segments.
[0026] For the purposes of the invention, the terms "polymer
segment" and "segment" denote a set of monomers linked together in
a linear and covalent manner, and belonging to a given type of
chemical composition, i.e. having specific overall physicochemical
properties, in particular as regards the salvation, the interaction
with a solid wall, a specific affinity toward certain molecules, or
a combination of these properties.
[0027] An example of a polymer segment for the purposes of the
invention is given by the sequence, within a copolymer, of monomers
that are all identical (homopolymer segment), or a copolymer that
has no significant composition correlation over distances of more
than a few monomers (segment of random copolymer type). The polymer
according to the invention is composed of several "different"
polymer segments. Two polymer segments that differ in their
chemical nature and/or their topology, i.e. the spatial
distribution of the segments relative to each other, for example
skeleton as opposed to side branch, are different for the purposes
of the invention.
[0028] According to a first preferred variant, the polymers
according to the invention are of the irregular block copolymer
type.
[0029] For the purposes of the invention, the term "block
copolymer" denotes a copolymer consisting of several polymer
segments linked together covalently, and belonging to at least two
different types of chemical composition. Thus, two adjacent polymer
segments within a linear block copolymer are necessarily of
significantly different chemical nature. The block copolymer is
defined by the fact that each of the segments comprises a
sufficient number of monomers to have within the separating medium
physicochemical properties, and in particular in terms of
salvation, that are comparable to those of a homopolymer of the
same composition and of the same size. This is in contrast with a
random copolymer, in which the various types of monomer follow each
other in an essentially random order, and give the chain locally
overall properties that are different from those of homopolymers of
each of the species under consideration. The size of the
homopolymer segments required to obtain this block nature may vary
as a function of the types of monomers and of the electrolyte, but
it is typically a few tens of atoms along the skeleton of said
segment. It should be noted that it is possible to make a block
copolymer within the meaning of the invention, in which all or some
of the segments themselves consist of a copolymer of random type,
insofar as it is possible to distinguish within said block
copolymer polymer segments of size and of difference in chemical
composition that are sufficient to give rise from one segment to
another to a significant variation in the physicochemical
properties, and in particular in the salvation. In particular, in
order to be considered as a "polymer segment" within the meaning of
the invention, a portion of polymer must comprise along its
skeleton at least 10 atoms.
[0030] According to one preferred mode of this variant, the polymer
according to the invention is of the irregular sequential block
copolymer type.
[0031] For the purposes of the invention, the term "sequential
block copolymer" means a block copolymer composed of polymer
segments belonging to at lest two different chemical types, linked
together in a linear manner.
[0032] According to a second preferred variant, the polymer
according to the invention is of the irregular comb polymer
type.
[0033] For the purposes of the invention, the term "comb polymer"
denotes a polymer having a linear skeleton of a certain chemical
nature, and polymer segments known as "side branches", of identical
or different chemical nature, which are also linear but
significantly shorter than the skeleton, and are covalently
attached to said skeleton via one of their ends. In a comb polymer,
the polymer segments constituting the skeleton and those
constituting the side branches differ in their topological nature.
If the polymer segments constituting the side branches of the comb
polymer and those constituting its skeleton also differ in their
chemical nature, the polymer simultaneously has the characteristic
of a "comb polymer" and that of a "block copolymer". Such polymers,
which are known as "comb copolymers", constitute a subset of comb
polymers and can, of course, be used in the context of the
invention.
[0034] Needless to say, the combined use of block copolymer(s) and
comb polymer(s) in a medium in accordance with the invention may be
envisaged.
[0035] The number of polymer segments of a given chemical or
topological type present in the polymers according to the invention
is understood as being an average value, it being understood that
it is always a matter of a population of a large number of
molecules having in said numbers a certain polydispersity.
[0036] In the present description and unless otherwise mentioned,
all the molecular masses and also all the averages for all the
chains or all the polymer segments, for instance the average
molecular mass, or the average number of atoms along the skeleton,
the number of junction points, or the average number of grafts in
the case of a comb polymer, are understood as being mass averages
within the usual meaning of polymer physics.
[0037] All the polymers under consideration according to the
invention, namely block copolymers or comb polymers, also have the
advantageous characteristic of being of irregular type, i.e. all
the segments of at least one type of chemical or topological nature
forming part of their composition have a polydispersity of at least
1.5 and preferably greater than 1.8.
[0038] The polydispersity of a type of polymer segment forming part
of the composition of a polymer according to the invention is
understood as being the average value of the molecular mass of said
segments, taken over all the segments of this type forming part of
the composition of said polymer (mass average within the usual
meaning of polymer physicochemistry).
[0039] One preferred variant of an irregular comb polymer consists
in displaying a polydispersity of the side branches of at least 1.5
and preferably greater than 1.8.
[0040] Another preferred variant of an irregular comb polymer
consists in displaying a polydispersity of the segments of the
skeleton included between two side branches of at least 1.5 and
preferably greater than 1.8.
[0041] In another preferred embodiment, the segments of each of the
types of chemical or topological nature forming part of the
composition of the polymers according to the invention have a
polydispersity of at least 1.5 and preferably greater than 1.8.
[0042] According to one preferred embodiment, the polydispersity of
the polymers according to the invention is greater than 1.5 and
preferably greater than 1.8.
[0043] The length and number of the different polymer segments
present in the comb polymers or the copolymers used in the media
according to the invention, and also the chemical nature thereof,
may vary significantly in the context of the invention, and the
properties of said media may thus be varied widely depending on the
desired application, as will be shown more specifically in the
description of the implementation examples.
[0044] According to one preferred embodiment, the polymers
according to the invention have a molecular mass (mass-average) of
greater than 50 000, preferably greater than 300 000, more
preferably greater than 1 000 000 and better still greater than 3
000 000.
[0045] According to one preferred embodiment, said polymers
according to the invention show within the separating medium
significant affinity for the walls of said channel.
[0046] One particularly preferred mode consists in presenting
within the polymer according to the invention at least one type of
polymer segment showing, within the separating medium, specific
affinity for the wall, and at least one type of polymer segment
showing in said medium less or no affinity for the wall.
[0047] The presence of polymer segments of this type allows the
medium according to the invention to reduce the adsorption of
species onto the walls of the channel and/or the
electroosmosis.
[0048] Specifically, one problem for all the methods involving
species within channels is the adsorption of said species onto the
walls of said channels. This problem is particularly exacerbated in
the case of small channels and biological macromolecules, the
latter often being amphiphilic. This phenomenon of adsorption onto
walls of species contained in the sample or the fluid has the
consequence of retarding certain analytes and of creating an
additional dispersion, and thus a loss of resolution, in the case
of analytical methods. This adsorption may also give rise to a
certain amount of contamination of the walls of the channel, which
may affect the fluids that it is desired to introduce thereafter
into said channel.
[0049] Another limitation, which more particularly relates to
electrokinetic separation methods, is electroosmosis, a movement as
a whole of the separating medium due to the presence of charges on
the walls of the capillary or the channel. Since this movement is
often variable over time and nonuniform, it is detrimental to the
reproducibility of the measurements and to the resolution. It is
caused by the charges that may be present on the surface of the
capillary due to its chemical structure, but may also be created or
increased by the adsorption, onto the wall, of charged species
initially contained in the samples to be separated, and in
particular proteins.
[0050] The polymers according to the invention of the type
containing polymer segments of at least one type showing within the
separating medium specific affinity for the wall, have, on account
of the presence of a plurality of segments of this type, and on
account of the relatively high average molecular mass of said
segments, a high adsorption energy, and thus reduce the
electroosmosis in a long-lasting manner. Moreover, since the
polymers according to the invention also comprise in their
structure polymer segments that show in said medium less or no
affinity for the wall, they avoid an excessively hydrophobic nature
that is harmful for resolution, and can more efficiently repel the
analytes from the walls.
[0051] Typically, types of polymer segments that show no affinity
for the wall consist of polymers that show good solubility in the
separating medium. However, there may be polymers which are soluble
in said medium, but which nevertheless show therein particular
affinity for a wall. When the separating medium is an aqueous
solution, segments with no affinity for the wall are typically
highly hydrophilic segments. On the other hand, segments with
affinity are relatively nonhydrophilic, or even hydrophobic.
Needless to say, other more specific types of affinity may be used,
depending on the nature of the wall and that of the separating
medium.
[0052] Copolymers that are optimized for performing the invention
are especially those in which all the segments that have specific
affinity for the wall represent between 2% and 80% by mass and
preferably between 5% and 30% of the total average molar mass of
said copolymers, or between 3% and 85% and preferably between 5%
and 50% of the total composition of the copolymers in terms of
number of moles of monomers.
[0053] Another preferred embodiment, which is particularly
advantageous when the analytes are biological macromolecules,
consists in using polymers according to the invention that also
show specific affinity for one or more analytes.
[0054] This affinity may be obtained by incorporating into the
structure of said polymers polymer segments capable of showing
specific affinity for certain analytes. Such polymer segments may
consist, for example, and in a nonexhaustive manner, of a
predetermined sequence of different monomers, for instance a
polynucleotide or a polypeptide. This affinity may also be obtained
by combining with the polymer according to the invention a native
or denatured protein, a protein fraction or a protein complex, or
alternatively an acidic or basic function, and/or a function of
Lewis acid or Lewis base type.
[0055] As illustrations of the various structures that may be
adopted by the copolymer according to the invention, mention may be
made most particularly of those in which all or some of said
copolymer is:
[0056] in the form of irregular sequential block copolymers. In
this case, one preferred variant consists in alternating, along the
polymer, segments with specific affinity for the wall, and segments
with reduced or no affinity for the wall. It may also be envisaged
to alternate, along the polymer, segments showing specific affinity
for certain analytes, and segments showing reduced or no affinity
for said analytes;
[0057] in the form of irregular comb copolymers. In this case, one
preferred variant is characterized in that said polymers are in the
form of comb polymers whose skeleton consists of several polymer
segments that show specific affinity with the wall, and the side
branches of which consist of polymer segments showing reduced or no
affinity for the wall, or comb polymers whose side branches consist
of polymer segments showing specific affinity for the wall, and
whose skeleton consists of polymer segments showing reduced or no
affinity for the wall. These polymers may also be in the form of
comb polymers, certain side branches of which consist of polymer
segments showing specific affinity for certain analytes, and the
skeleton of which consists of polymer segments showing reduced or
no affinity for these analytes.
[0058] Needless to say, systems in which several types of preferred
variants above are combined together, either by combining polymer
segments of more than two different types, or in the form of a
mixture of different copolymers, also fall within the scope of the
invention. It is thus possible, for example, to combine within a
copolymer according to the invention polymer segments showing
affinity for the wall, polymer segments or groups showing specific
affinity for certain analytes, and polymer segments showing no
specific affinity either for the walls or for the analytes. It is
also possible, again by way of example, to combine in a medium
according to the invention block copolymers comprising polymer
segments showing affinity for the wall and polymer segments showing
no specific affinity either for the walls or for the analytes, and
polymers comprising polymer segments or groups showing specific
affinity for certain analytes, and polymer segments showing no
specific affinity either for the walls or for the analytes.
[0059] In one preferred mode of the invention, all of the polymer
segments of a given type of chemical or topological nature have on
average along their skeleton a number of atoms of greater than 75,
and more preferably greater than 210, or have a molecular mass of
greater than 1 500 and preferably greater than 4 500.
[0060] According to an even more preferred embodiment, the various
types of segments have along their skeleton an average number of
atoms of greater than 75, and more preferably greater than 210, or
have a molecular mass of greater than 1 500 and preferably greater
than 4 500.
[0061] According to one preferred embodiment, the separating medium
consists of a liquid in which at least one polymer in accordance
with the invention is dissolved to a proportion of from 0.1% to 20%
and preferably from 1% to 6% by weight.
[0062] It is particularly advantageous for implementing the
invention to use block copolymers or comb homopolymers in which at
least one of the types of segments consists of a polymer chosen
from polyethers, polyesters, for instance polyglycolic acid,
soluble random homopolymers and copolymers of the polyoxyalkylene
type, for instance polyoxypropylene, polyoxybutylene or
polyoxyethylene, polysaccharides, polyvinyl alcohol,
polyvinylpyrrolidone, polyurethanes, polyamides, polysulfonamides,
polysulfoxides, polyoxazoline, polystyrene sulfonate, and
substituted or unsubstituted acrylamide, methacrylamide and allyl
polymers and copolymers.
[0063] As representatives of the types of polymer segments showing,
in an aqueous separating medium, little or no affinity with the
walls, mention may be made most particularly of polyacrylamide and
polyacrylic acid, polyacryloylaminopropanol, water-soluble acrylic
and allylic polymers and copolymers, dextran, polyethylene glycol,
polysaccharides and various cellulose derivatives such as
hydroxyethylcellulose, methylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose or methylcellulose, polyvinyl alcohol,
polyurethanes, polyamides, polysulfonamides, polysulfoxides,
polyoxazoline, polystyrene sulfonate, and also polymers bearing
hydroxyl groups, and all the random copolymers of the derivatives
mentioned above.
[0064] Needless to say, other polymer segments that are soluble in
the separating medium may be used according to the invention, as a
function of the nature of said fluid and of that of the walls of
the channel, the particular application and the ease of introducing
them into a block polymer of the desired structure.
[0065] As representatives of the polymer segments, which may or may
not be soluble in aqueous solvents, and which may show therein
particular affinity for the walls, mention may be made of
dimethylacrylamide, acrylamides N-substituted with alkyl functions,
acrylamides N,N-disubstituted with alkyl functions, allyl glycidyl
ether, copolymers of the above acrylic derivatives with each other
or with other acrylic derivatives, alkanes, fluoro derivatives,
silanes, fluorosilanes, polyvinyl alcohol, polymers and copolymers
involving oxazoline derivatives, and also in general polymers that
have a combination of carbon-carbon bonds, ether-oxide functions
and epoxide functions, and also all the random copolymers of these
compounds.
[0066] Many types of polymer segments may be chosen to make up the
polymer segments constituting a polymer according to the invention,
as a function of the envisaged electrolyte, from all the types of
polymers known to those skilled in the art, in particular from
those soluble in aqueous medium. Reference may thus be made to the
book "Polymer Handbook" Brandrupt & Immergut, John Wiley, New
York.
[0067] The polymers according to the invention may be natural or
synthetic. According to one preferred variant for the variety and
control that it allows with regard to the microstructure, the
polymers according to the invention are synthetic polymers.
[0068] The following are most particularly suitable for the
invention:
[0069] copolymers of the comb copolymer type, the skeleton of which
is of dextran, acrylamide, acrylic acid, acryloylaminoethanol or
(N,N)-dimethylacrylamide type and onto which are grafted side
segments of acrylamide, substituted acrylamide or
(N,N)-dimethylacrylamide (DMA) type, or of the DMA/allyl glycidyl
ether (AGE) copolymer type, or alternatively of homopolymer or
copolymer of oxazoline or of oxazoline derivatives;
[0070] non-thermosensitive copolymers of the irregular sequential
block copolymer type having along their skeleton an alternation of
segments of polyoxyethylene type and of segments of
polyoxypropylene type, or an alternation of segments of
polyoxyethylene type and of segments of polyoxybutylene type, or
more generally an alternation of segments of polyethylene and of
segments of polyether type that are appreciably more hydrophobic
than polyoxyethylene;
[0071] copolymers of the irregular sequential block copolymer type
having along their skeleton an alternation of segments of
acrylamide, acrylic acid, acryloylaminoethanol or
dimethylacrylamide type, on the one hand, and segments of
(N,N)-dimethylacrylamide (DMA) type, or of DMA/allyl glycidyl ether
(AGE) copolymer type, or alternatively of homopolymer or copolymer
of oxazoline or of oxazoline derivatives;
[0072] polymers of the irregular comb polymer type, the skeleton of
which is of agarose, acrylamide, substituted acrylamide, acrylic
acid, acryloylaminoethanol, dimethylacrylamide (DMA), or allyl
glycidyl ether (AGE) polymer type, of DMA/AGE random copolymer
type, of oxazoline and oxazoline derivative, of dextran, of
methylcellulose, of hydroxyethylcellulose, of modified cellulose,
of polysaccharide or of ether oxide type, and onto which are
grafted side segments of agarose, acrylamide, substituted
acrylamide, acrylic acid, acryloylaminoethanol, dimethylacrylamide
(DMA), or allyl glycidyl ether (AGE) polymer type, of DMA/AGE
random copolymer type, of oxazoline and oxazoline derivative, of
dextran, of methylcellulose, of hydroxyethylcellulose, of modified
cellulose, of polysaccharide or of ether oxide type;
[0073] copolymers of the irregular comb copolymer type, the
skeleton of which is of the acrylamide, substituted acrylamide,
acrylic acid, acryloylaminoethanol, dimethylacrylamide (DMA), or
allyl glycidyl ether (AGE) polymer type, of DMA/AGE random
copolymer type, of oxazoline and oxazoline derivative, of dextran,
of agarose, of methylcellulose, of hydroxyethylcellulose, of
modified cellulose, of polysaccharide or of ether oxide type, and
bears short-chain hydrophobic side segments such as alkyl chains,
aromatic derivatives, fluoroalkyls, silanes or fluorosilanes.
[0074] It should also be noted that, in most applications, it is
preferable to use a polymer according to the invention that is
essentially neutral. However, it may be useful for certain
applications, and in particular to avoid the adsorption of species
containing both charges and hydrophobic portions, to select a
polymer according to the invention that is deliberately charged,
preferably opposite in charge to that of said species.
[0075] As regards the preparation of the copolymers used according
to the invention, it may be carried out by any conventional
polymerization or copolymerization technique. The choice of
preparation method is generally made by taking into account the
structure desired for the copolymer, i.e. comb or linear structure,
and the chemical nature of the various blocks of which it is
made.
[0076] As representatives of these preparation variants, mention
may be made most particularly of processes according to which said
copolymers are obtained by:
[0077] polycondensation, ionic or free-radical polymerization or
copolymerization of identical or different monomers, of identical
or different macromonomers, or of a mixture of identical or
different monomers and macromonomers, or
[0078] by grafting several polymer segments onto a linear or
branched polymer skeleton of identical or different chemical
nature.
[0079] Preferably, all or some of the copolymers used according to
the invention are obtained by
[0080] a: copolymerization of monomers and macromonomers comprising
a reactive function at at least one of their ends, or
[0081] b: copolymerization of macromonomers comprising at least one
reactive function in their structure.
[0082] For the purposes of the invention, the term "reactive
function" means a group that allows the molecule bearing this group
to be incorporated into the macromolecule during the
copolymerization reaction without interrupting said
copolymerization.
[0083] With the aid of the rules and preferred modes listed above,
a person skilled in the art is capable of preparing the copolymers
in accordance with the invention, by adapting the structure, the
nature and the mode of preparation of said polymers as a function
of the desired separation properties for one application or
another.
[0084] A subject of the present invention is also a process for
separating, analyzing and/or identifying species contained in a
sample, characterized by performing the following steps:
[0085] a/ filling the channel of a separating device with
separating medium according to the invention,
[0086] b/ introducing said sample containing said species into one
end of said channel,
[0087] c/ applying an external field intended to move certain
species contained in the sample, especially an electric field,
and
[0088] d/ recovering or detecting the passage of said species at a
point along the channel that is different from the point of
introduction of the sample.
[0089] In one preferred variant, it is not necessary to change the
temperature between the capillary filling stage and the analysis
stage.
[0090] Depending on the particular applications, the separating
medium may contain, besides the polymers according to the
invention, other elements, and in particular components that
interact with the species or the walls. Many elements of this type
are known to those skilled in the art.
[0091] In the present case, it is possible to combine in the
separating medium polymers of the irregular block copolymer type,
and other polymers capable of interacting with analytes either by
steric interaction or by affinity, in order to improve the
performance qualities compared with those obtained with the polymer
according to the invention used alone. In this case, polymers that
are more particularly preferred according to the invention are
those having a mass fraction of polymer segments showing specific
affinity for the wall that is greater than when these polymers are
used alone. This fraction may be between 20% and 80%.
[0092] A subject of the present invention is also the use of a
separating medium according to the invention for separating,
purifying, filtering or analyzing species chosen from molecular or
macromolecular species, and in particular biological
macromolecules, for instance nucleic acids (DNA, RNA or
oligonucleotides), nucleic acid analogs obtained by synthesis or
chemical modification, proteins, polypeptides, glycopeptides and
polysaccharides, organic molecules, synthetic macromolecules or
particles such as mineral particles, latices, cells or
organelles.
[0093] In the case of electrophoresis analysis methods, the
invention is particularly useful for DNA sequencing, for which it
allows minimum bandwidths to be obtained. It is also particularly
favorable for separating proteins, proteoglycans, or cells, for
which the problems of adsorption onto the wall are a particular
handicap and particularly difficult to solve.
[0094] Advantageously, the claimed medium may be used in a channel
of which at least one dimension is of submillimetric size.
[0095] As regards the apparatus, the claimed medium is particularly
advantageous for microfluidic systems, since it makes it possible,
by means of an optimum choice of the various types of blocks within
the polymers, to combine blocks that show good affinity for the
surface of the channel in order to obtain a long-lasting treatment,
and blocks that show good repulsion for the species to be
separated, irrespective of said species and of the chemical nature
of said channel.
[0096] The media according to the invention and the separation
methods using these media are particularly advantageous for
electrophoretic separation and diagnostic applications, gene
typing, and large-throughput screening, quality control, or for
detecting the presence of genetically modified organisms in a
product.
[0097] In point of fact, the polymers of which the separating
medium under consideration in the context of the present invention
is composed are found to be advantageous in several respects.
[0098] Firstly, their capacity to display "block polymer" nature
allows them to combine properties belonging to polymers of
different chemical nature, and that cannot always be united in a
homopolymer or a random copolymer. They thus make it possible to
more flexibly adapt the chemical nature of the separating medium,
firstly as a function of the species to be separated, and secondly
as a function of the chemical nature of the channels in which the
separation is performed. They are thus particularly advantageous
both in applications using channels consisting of polymers or
elastomers such as PDMS (polydimethylsiloxane), PMMA (polymethyl
methyacrylate), polycarbonate, polyethylene, polypropylene,
polyethylene terephthalate or polyimide, or of mineral materials
such as glass, ceramics, silicon, stainless steel or titanium, and
in more traditional applications using channels whose walls are
made of fused silica.
[0099] Compared with the block copolymers of the prior art, the
polymers according to the invention also show superior performance
qualities in terms of resolution, which is most probably associated
with their irregular nature, i.e. the polydispersity of the polymer
segments forming part of the polymers according to the invention.
This characteristic is particularly surprising, since the set of
block copolymers used in the prior art deliberately involves
copolymers containing regularly spaced segments and/or having a
selected and essentially uniform length (i.e. low polydispersity).
This polydispersity of the segments, in the polymers according to
the invention, also shows advantages in terms of cost and
flexibility in formulating, since polymers comprising such
polymolecular segments are not only more efficient, but also easier
to prepare. In particular, they may be prepared with high molecular
masses.
[0100] In the applications for which a reduction of electroosmosis
or interaction of species with the wall is desired, the polymers
according to the invention have, on account of the presence in
their structure of a large number of polymer segments that show
significant affinity for the wall, high adsorption energy and can
thus reduce the electroosmosis and the adsorption of species in a
long-lasting manner.
[0101] Finally, it is also very likely that the combination of a
linear skeleton and of a plurality of junction points gives the
separating media according to the invention some of the properties
of gels at the local scale, which is beneficial in terms of
separation efficiency, while at the same time conserving them at
the large scale, and in particular as regards the flow properties,
properties that are comparable with those of linear polymers.
[0102] The figures and examples given below are presented as
nonlimiting illustrations of the present invention.
FIGURES
[0103] FIG. 1: Example of diagrammatic configuration 1a: of an
irregular sequential block copolymer; 1b: of an irregular comb
polymer; 1c: of an irregular comb copolymer. The bold lines
correspond to one type of chemical nature, and the fine lines to
another type of chemical nature.
[0104] FIG. 2: Control electrophoregram representing the separation
of the Pharmacia Biotech 50-500 bp sizer, obtained at 50.degree. C.
in an ABI 310 machine (Perkin Elmer), using an untreated capillary
and, as separating medium, a 100 mM Na TAPS, 2 mM EDTA, 7M urea
buffer, in which is dissolved 5% of a commercial homopolymer of the
polyacrylamide type (molecular mass 700 000-1 000 000); the DNA
sizes corresponding to the various peaks are indicated on the
diagram, as number of bases.
[0105] FIG. 3: Control electrophoregram representing a separation
under conditions identical to those of FIG. 2, with a "POP6"
commercial separating medium from PE Biosystems. The DNA sizes
corresponding to the various peaks are indicated on the diagram, as
number of bases.
[0106] FIG. 4: Electrophoregram representing a separation under
conditions identical to those of FIG. 2, with a 100 mM Na TAPS, 2
mM EDTA, 7M urea separating medium, in which is dissolved 5% of
P(AM-PDMA)-2 comb copolymer described in Example 2. The DNA sizes
corresponding to the various peaks are indicated on the diagram, as
number of bases.
[0107] FIG. 5: Comparison of the resolution calculated between
peaks differing from one base to 500 bases, obtained at 50.degree.
C. in an ABI 310 machine (Perkin-Elmer), using as separating
medium:
[0108] a: a "Pop6" commercial separating medium from PE
Biosystems,
[0109] b: a 50 mM Na TAPS, 2 mM EDTA, 7M urea buffer, in which is
dissolved 5% of linear acrylamide (molecular mass 700 000.about.1
000 000),
[0110] c: the same buffer, in which is dissolved 5% of irregular
block copolymer according to the invention P(AM-PDMA)-2 described
in Example 2.
[0111] FIG. 6: Viscosity of solutions at 3% of linear acrylamide
(LPA) and of the copolymers according to the invention
poly(AM-PDMA)-1, prepared according to Example 2, poly(AM-PDMA)-2,
prepared according to Example 4, and poly(AM-PDMA)-3, prepared
according to Example 5.
[0112] FIG. 7: Resolutions obtained during the electrophoretic
separation of DNA, in solutions of linear acrylamide (LPA), of
commercial polymer (POP5), and of the copolymers according to the
invention poly(AM-PDMA)-1, prepared according to Example 2,
poly(AM-PDMA)-2, prepared according to Example 4, and
poly(AM-PDMA)-3, prepared according to Example 5, at 3% and 5%.
EXAMPLE 1
[0113] Preparation of a functionalized PDMA macromonomer with a
molecular mass in the region of 10 000, for the preparation of
copolymers in accordance with the invention.
[0114] 1) Polymerization of PDMA
[0115] The free-radical polymerization of N,N-dimethylacrylamide
(DMA) is performed in pure water. The initiator is a redox couple
for which the oxidizing agent is potassium persulfate
K.sub.2S.sub.2O.sub.8 (KPS) and the reducing agent is
aminoethanethiol AET.HCl. The initiation reaction is:
[0116] K.sub.2S.sub.2O.sub.8+2Cl.sup.-,
NH.sub.3.sup.+--CH.sub.2CH.sub.2---
SH.fwdarw.2KHSO.sub.4+2Cl.sup.-,
HN.sub.3.sup.+--CH.sub.2--CH.sub.2--S.sup- .+
[0117] AET.HCl also acts as transfer agent, which allows the chain
length to be controlled.
[0118] Procedure
[0119] 0.18 mol of DMA and 200 ml of water are placed in a 500 ml
three-necked flask on which is mounted a condenser, and equipped
with a nitrogen inlet device. The mixture is then stirred and
heated to 29.degree. C. with a water bath. Sparging with nitrogen
is commenced. After 45 minutes, 0.61 g of AET.HCl (0.0054 mol)
predissolved in 20 ml of water is added, followed by addition of
0.0018 mol of potassium persulfate (KPS) dissolved in a minimum
amount of water. The mixture is stirred for 3 hours. The solution
is then concentrated and then freeze-dried.
[0120] To isolate the polymer, a precipitation is performed
according to the following procedure:
[0121] The solid obtained is redissolved in 100 ml of methanol. The
hydrochloride present is neutralized by adding 0.0054 mol of KOH
(i.e. 0.30 g dissolved in about 25 ml of methanol) incorporated
dropwise into the solution. The salt formed, KCl, precipitates and
is extracted by filtration. The filtrate thus recovered is
concentrated and then poured dropwise into 4 liters of ether. The
precipitated polymer is recovered by filtration through a No. 4
sinter funnel. The solid is then dried under vane-pump vacuum. The
mass yield is about 50%.
[0122] The above protocol leads to an amino polymer known as "PDMA"
and corresponds to initiator/monomer ratios Ro=0.03 and Ao=0.01, in
which:
[0123] Ro=[R--SH]/[NIPAM] and Ao=[KPS]/[NIPAM].
[0124] 2) Modification of the Amino PDMA
[0125] The PNIPAM macromolecules synthesized contain amine
functions at the chain ends, these chains originating from the
initiator aminoethanethiol AET.HCl.
[0126] By reaction of the amine function with acrylic acid, a vinyl
double bond is attached to the chain end according to the following
reaction scheme: 1
[0127] Procedure:
[0128] 50 ml of methylene chloride, 1.5 g of acrylic acid (0.021
mol), 9 g of PDMA and 4.3 g of dicyclohexylcarbodiimide (DCCI)
(0.021 mol) are placed in a 100 ml beaker.
[0129] The reaction medium is stirred for one hour. Since the
acrylic acid is in large excess relative to the PDMA (the amount of
acrylic acid is about twenty times that of the PDMA), all the amino
functions have been modified. The mixture is then filtered through
a No. 4 sinter funnel to remove the precipitated dicyclohexylurea,
the by-product resulting from the conversion of the DCCI. The
purification is performed by precipitation from ether.
[0130] A macromonomer PDMA-1 bearing an allyl function at the chain
end is thus obtained with a mass yield of about 70%.
[0131] The average molar mass and the polydispersity of the
macromonomers thus prepared, measured by SEC (steric exclusion
chromatography), are of the order of 15 000 and 2,
respectively.
EXAMPLE 2
[0132] Preparation of a copolymer P(AM-PDMA)-1 with an acrylamide
skeleton and PDMA grafts, of molecular mass 1 500 kdalton.
[0133] The copolymerization of amino PDMA (0.4 g) and of acrylamide
(2.8 g) is performed for 4 hours in 50 ml of water at room
temperature, while degassing vigorously with argon. The initiator
used is the redox couple of ammonium persulfate
((NH.sub.4).sub.2S.sub.2O.sub.8) [0.075 mol % of the amount of
monomers]/sodium metabisulfite (Na.sub.2S.sub.2O.sub.5) (0.0225 mol
% of the amount of monomers). The resulting copolymer is purified
by precipitation from acetone and dried under vacuum. Its molecular
mass is 1 500 kdalton, and its polydispersity Mw/Mn is about 2. The
degree of incorporation of macromonomer, measured by proton NMR, is
about 6%, which corresponds to an average number of side branches
on the skeleton of about 6.
[0134] On account of the free-radical polymerization method used,
the macromonomers constituting the side chains are incorporated
into the polymer chain at random positions determined by chance by
the collisions between molecules (random distribution). This
polymerization method leads to a distribution of the molecular
masses of the polymer segments of the skeleton between two side
branches of approximately exponential shape, and thus to
polydispersities of said polymer segments of the skeleton that are
largely superior to 1.8.
EXAMPLE 3
[0135] Separation properties obtained for single-stranded DNA
(50-500 bp sizer, Pharmacia Biotech) at 50.degree. C. in an ABI 310
machine (Perkin-Elmer), in a 100 mM Na TAPS, 2 mM EDTA, 7 M urea
buffer, in various separation media. It is observed visually (FIG.
4) and more quantitatively by means of the resolution measurements
(FIG. 5), that the separating medium according to the invention
P(AM-PDMA)-2 improves the resolution relative to the same polymer
skeleton not bearing side branches (PAM, FIGS. 2 and 5), but also
relative to a commercial product based on linear PDMA (POP6, FIGS.
3 and 5). The separation time is also reduced, which is an
additional advantage of the media according to the invention. It
thus appears, surprisingly but beneficially, that this polymer
according to the invention which comprises a large fraction of
acrylamide, and a smaller fraction of PDMA, has, on account of the
particular arrangement of said fractions and of the presence of
junction points that characterize the invention, properties that
are superior to those of each of said components in homopolymer
form.
EXAMPLE 4
[0136] Preparation of a copolymer P(AM-PDMA)-2 containing an
acrylamide skeleton and PDMA grafts, of molecular mass about 3 000
kdalton
[0137] The preparation is identical to that described in example 2,
except for the concentration of ((NH.sub.4).sub.2S.sub.2O.sub.8)
[0.1 mol % instead of 0.075 mol % of the amount of monomers] and of
(Na.sub.2S.sub.2O.sub.5) (0.015 mol % instead of 0.0225 mol % of
the amount of monomers). The viscosity, presented in FIG. 6, makes
it possible to evaluate the molecular mass, of about 3 000 kdalton,
starting from that of the p(AM-PDMA)-1, using the cubic dependence
of the viscosity as a function of the molecular mass for
interlocked polymers.
EXAMPLE 5
[0138] Preparation of a copolymer P(AM-PDMA)-3 bearing PDMA grafts,
of molecular mass about 30 000.
[0139] In a first stage, the macromonomer of molecular mass 30 000
is prepared as described in example 1, with the exception of the
ratio Ro, which is set at 0.015 instead of 0.03. This macromonomer
is then polymerized with acrylamide, according to the protocol
described in example 4.
EXAMPLE 6
[0140] Measurement of the viscosity of 3% solutions obtained with
the polymers described in examples 2, 4 and 5, and also with a
linear acrylamide homopolymer. In this example, each of the
polymers was introduced at a rate of 3 g/100 ml into purified water
(MilliQ). The viscosity of each of the corresponding solutions was
measured on a Brookfield DV3 cone-plate rheometer run by the
Rheocalc software (Sodexim, Muizon, F). The shear rate selected is
10 (1/s) for a temperature gradient of 1.degree. C. per minute. It
is observed in FIG. 6 that the copolymers according to the
invention have no thermosensitive nature (their viscosity shows a
small and uniform decrease with temperature), and a moderate
viscosity. It is also observed that the structure and properties of
the copolymers can be varied by controlling the polymerization
conditions.
EXAMPLE 7
[0141] Electrophoretic separations of single-stranded DNA
fragments, in separating media according to the invention based on
copolymers described in examples 2, 4 and 5, and, for comparative
purposes, in linear polyacrylamide (LPA) and the commercial
separating medium "POP5" (Applied Biosystems). The separation
conditions are identical to those of example 4, with the exception
of the sample, a "sizer" of 100 to 1 500 bases (BioVentures, USA).
It is observed in FIG. 7 that in the range that is most
advantageous for sequencing (fragment size 600 to 1 000), the media
based on copolymers according to the invention, in particular those
corresponding to a mass concentration in the separating medium of
3%, lead to a resolution that is markedly superior to that obtained
with the polymers of the prior art. Considering that a resolution
of the order of 0.3 to 0.5 is sufficient to sequence DNA to within
one base, the media according to the invention should allow reading
lengths of greater than 800 bases.
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