U.S. patent application number 10/078401 was filed with the patent office on 2002-06-27 for composition for use as an electrophoretic medium.
Invention is credited to Roy, Dominique, Sevigny, Pierre.
Application Number | 20020079222 10/078401 |
Document ID | / |
Family ID | 23531266 |
Filed Date | 2002-06-27 |
United States Patent
Application |
20020079222 |
Kind Code |
A1 |
Sevigny, Pierre ; et
al. |
June 27, 2002 |
Composition for use as an electrophoretic medium
Abstract
The present invention is concerned with a disposable
electrophoresis cassette particularly suitable for pre-cast
polyacrylamide gels for protein and nucleic acid electrophoresis.
The invention also comprises a support plate for the said cassette
that acts as a heat sink and provides a more uniform migration
front in operation since the temperature of the gel is
substantially the same during electrophoresis operation. Also
disclosed is a novel comb element for filling the cassette, and a
novel method therefor.
Inventors: |
Sevigny, Pierre; (Montreal,
CA) ; Roy, Dominique; (Montreal, CA) |
Correspondence
Address: |
OGILVY RENAULT
1981 MCGILL COLLEGE AVENUE
SUITE 1600
MONTREAL
QC
H3A2Y3
CA
|
Family ID: |
23531266 |
Appl. No.: |
10/078401 |
Filed: |
February 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10078401 |
Feb 21, 2002 |
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09387758 |
Sep 1, 1999 |
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6379519 |
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Current U.S.
Class: |
204/469 ;
204/456; 204/466; 204/467; 204/470; 204/606; 204/616; 204/618;
204/619; 204/620 |
Current CPC
Class: |
B29C 51/10 20130101;
B29C 35/16 20130101; B29C 2791/006 20130101; B29C 2793/0018
20130101; G01N 27/44747 20130101; B29C 51/30 20130101; G01N
27/44704 20130101; B29C 51/428 20130101; B29C 51/082 20130101 |
Class at
Publication: |
204/469 ;
204/467; 204/466; 204/456; 204/606; 204/616; 204/618; 204/619;
204/620; 204/470 |
International
Class: |
G01N 027/26; G01N
027/447 |
Claims
What is claimed is:
1. A composition for use as an electrophoretic medium comprising:
an electrophoretic gel; a polymerization initiator; a buffer; and
an adhesive agent allowing adhesion of polymerized electrophoretic
medium onto a surface of an thermoplastic electrophoretic
cassette.
2. A composition according to claim 1 wherein the gel comprises
acrylamide gel, optionally cross-linked, agarose gel or starch gel;
the polymerization initiator comprises ammonium persulfate and
N,N,N,N-tetramethylethylenediamine (TEMED);
4-dimethylaminopropionitrile; 1-hydroxycyclohexyl phenyl ketone;
2,2-diethoxy-acetophenone; 2,2-dimethoxy-2-phenylacetophenone;
2',4'-dimethoxyacetophenone; 2-hydroxy-2-methyl-1-propiophenone;
2-hydroxy-2-methyl-1-phenylpropan-1-o- ne, and mixtures thereof;
the buffer comprises tris-borate EDTA, tris-acetate-EDTA,
tris-glycine, tricine, or mixtures thereof; and the adhesive agent
comprises at least one tetra-substituted silicon derivative wherein
the substituents are the same or different and comprise a straight
or branched alkyl, alkoxy, ketone, ester or amide each comprising
from 1 to 8 carbon atoms, amino, halogen, cyano or hydroxy.
Description
[0001] This application is a divisional of the application Ser. No.
09/387,758 filed on Sep. 1, 1999.
BACKGROUND OF THE INVENTION
[0002] (a) Field of the Invention
[0003] The present invention is concerned with a disposable
electrophoresis cassette particularly suitable for pre-cast
electrophoretic media for protein and nucleic acid electrophoresis.
The invention also comprises a support plate for the said cassette
to ensure proper rigidity thereof into the electrophoresis
apparatus, and acting as a heat sink, thus providing a more uniform
migration front in operation because the temperature of the medium
is substantially even anywhere inside the cassette during
electrophoresis operation. The invention also comprises a comb for
injection of electrophoresis medium into the cassette, as well as
the corresponding method of injection.
[0004] (b) Background of the Invention
[0005] Electrophoresis is a well known separation technique that
requires the application of electrical current at both poles of a
cassette or plate to force samples through an electrophoretic
medium that acts as a molecular sieve. The application of a
difference of potential between the upper section and the lower
section of the cassette assumes the creation of two areas sealed
from each other. Because current is transmitted via two separate
buffer reservoirs, it is necessary to apply a pressure or force on
the cassette so that the seals properly operate. It is therefore
imperative that the whole system, including the cassette, possess
some rigidity.
[0006] Conventional electrophoresis cassettes are made of two glass
plates spaced apart with plastic spacers or tongues to create a
space therebetween while ensuring that the sides of the assembly
are properly sealed. Importantly, the spacers must not conduct
electrical current. The assembly is generally maintained together
with clamps, and it is often necessary to reinforce the seals with
hot agar. When the gel is cast into the cassette, a comb element is
introduced at one end of the assembly to create one or more
reservoirs or wells thereafter wherein the sample(s) will be
received later. The shape of the comb may comprise various numbers
and sizes of reservoirs, depending on the application required and
the size of the cassette. For example, a preparation gel
necessitate less reservoirs, while an analytical gel will require
more reservoirs and the width thereof will depend on the resolution
desired.
[0007] However, such assemblies have several drawbacks and
limitations. The assembling operation requires dexterity and is a
time-consuming operation, because it is done manually. The plates
are conventionally made of glass, and thus must be handled with
care. Further, they must be carefully cleaned to obtain good
results. Finally, manipulation of acrylamide gel, a commonly used
electrophoretic medium, represents a long-term danger for the
health of operators since such gel is highly toxic.
[0008] More recently, to simplify the assembling work of operators
and reduce poisoning and manipulation hazards, pre-cast cassettes
already containing the gel have been made available commercially.
The cassettes comprise an acrylamide gel, and a comb is provided at
one extremity thereof. However, the cost of these cassettes is
prohibitive, and demolding thereof, for visualization of the
results, is a delicate and complicated procedure. In addition, the
comb is produced by injection molding, and is used to form the
wells or reservoirs in the gel. They generally represent an
important part of the total cost of the cassette.
[0009] To be economically feasible and capable of supporting,
without substantial bending, the mechanical forces applied thereon,
cassettes containing pre-cast electrophoresis medium, must be rigid
enough and made of a material economically sound and preferably
recyclable, such as for example thermoplastic materials like
polymethylmethacrylate (PMMA). However, conventionally, in order to
be sufficiently rigid, the plates must be relatively thick. Two
obvious problems therefore become apparent: a) the amount of
thermoplastic material required is significant, thus increasing the
cost, which is not suitable for a disposable device; and b)
maintaining the gel at an appropriate operating temperature is
complicated, because the thick walls of the thermoplastic material
act as a dielectric material. Thicker plastic walls also affect the
diffusion of the heat generated during the electrophoretic process,
creating temperature gradients within the electrophoresis medium,
and non-uniform migration of the samples analyzed.
[0010] Conventional processes for filling the cassettes are
generally standard, irrespective of the electrophoretic medium.
Typically, a gel comprising a mixture of acrylamide and
bis-acrylamide, a buffer like tris-borate ethylenediamine (EDTA),
tris-acetate-EDTA, tris-glycine, tricine, and a polymerization
initiator are injected or cast into the cassette. Some of these
products are neurotoxic and/or irritant, and must therefore be
handled with extreme care. A laboratory pipette or a pump can be
used to fill the cassette from the top with the liquid medium. Once
the cassette is filled, a comb closes the top of the cassette. The
comb has a design such that it contains one or more teeth forming
reservoirs in the gel wherein the sample will be placed later.
After polymerization of the medium, the comb is removed, as well as
a separator present in the lower portion of the cassette. The
cassette is then placed in an electrophoresis apparatus wherein the
lower and upper portions of the gel will be in contact with two
independent buffer solutions. The samples are then introduced in
the reservoirs, and current is applied to separate the various
components of each sample. After completion of the separation, the
medium is removed from the cassette for further processing, i.e.,
coloration, photograph and analysis.
[0011] Again, such system and procedure have various major
drawbacks and limitations. As stated above, manual filling of the
cassette requires great care and dexterity, not to mention exposure
of the operator to toxic chemicals. Further, undesirable bubbles
often form during filling, and installation of the comb after
filling may also create bubbles at the bottom of the teeth. Such
air bubbles must be avoided at all times, since they interfere
significantly with the samples migrating in the polymerized gel
during the electrophoresis procedure.
[0012] Pre-cast gels have been marketed recently, but have not been
able to overcome other problems mentioned above for cassettes
containing the same, such as prohibitive costs. One of the main
reason is that the cassettes are obtained by injection molding,
which is a costly and relatively slow process because of the
significant amount of plastic required for injection, the cost of
the plastic material itself, and the time necessary to allow
complete cooling of the cassette thus obtained. In addition,
because the cassettes are made of a thermoplastic material, gel
polymerization is greatly affected and slowed down because the
polymer absorbs free radicals generated by the chain reaction of
the polymerization. As a result, the polymerized electrophoretic
medium does not "stick" do the cassette inner surfaces. An
expensive coating layer or overlay must therefore be applied on the
thermoplastic material surfaces to minimize this problem and ensure
proper polymerization quality and speed.
[0013] The electrophoresis operation necessitates the application
of a voltage across the gel that generates heat that must be
somehow dissipated. During the heat dissipation process, if the
temperature of the gel is not uniform, it causes distortion in the
separated protein or polynucleic acid bands. Such heat is therefore
a critical problem because it limits the rate at which gels can be
run. Increasing temperatures reduces the resistance and increases
current at a given voltage. Although the net effect is a shorter
run, excessive temperature can lead to undesirable band broadening.
It is therefore preferable to run at a higher voltage and a
constant lower temperature.
SUMMARY OF THE INVENTION
[0014] The present invention is concerned with an electrophoresis
cassette comprising:
[0015] first and second thermoformed surfaces comprising their
edges in hermetic contact with each other and defining a volume
therebetween for receiving an electrophoretic medium;
[0016] either of first or second surface comprising at least one
opened reservoir molded therein at one end; and
[0017] either of first or second surface further comprising at
least one aperture at an opposite end thereof.
[0018] In a preferred embodiment, the cassette comprises a male
member having a structure complementary to that of the at least one
reservoir, the male member being adapted to be removably inserted
therein and further comprising:
[0019] an inlet and an outlet for injecting the electrophoretic
medium therethrough and into the volume defined by the first and
second surfaces; and
[0020] at least one opening for discharge of the excess of the
electrophoretic medium.
[0021] In a second aspect of the invention, there is provided a
comb for an electrophoretic cassette adapted to be removably
inserted into the cassette and allowing injection therein of an
electrophoretic medium and discharge of excess of electrophoretic
medium therefrom, comprising at least one inlet and one outlet for
injection of the electrophoretic medium into the cassette, the
outlet extending longitudinally on a first side of at least one
tooth.
[0022] In a third aspect of the invention, there is provided an
electrophoresis assembly comprising:
[0023] a cassette as defined above;
[0024] an electrophoretic medium comprised in the volume; and
[0025] a support comprising a frame adapted to receive the
cassette, the support further comprising a plurality of recesses
forming corresponding channels between a cassette surface and the
support, thus allowing passage of a coolant therein, whereby the
temperature of the medium is substantially the same within the
volume.
[0026] In a fourth aspect of the invention, there is provided a
method for casting an electrophoretic medium into a cassette,
comprising the steps of:
[0027] sealing any aperture present in the cassette;
[0028] injecting the electrophoretic medium into the inlet of the
comb defined above, the medium penetrating into the volume through
the comb; and
[0029] continuing injection of the medium until all the volume is
filled and an excess thereof is discharged from at least one
opening of the comb.
[0030] Finally, in a fifth aspect of the invention, there is
provided a composition for use as an electrophoretic medium
comprising:
[0031] an electrophoretic gel;
[0032] a polymerization initiator;
[0033] a buffer; and
[0034] an adhesive agent allowing adhesion of polymerized
electrophoretic medium onto a surface of an thermoplastic
electrophoretic cassette.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 illustrates a cassette and corresponding support
plate in accordance with the present invention;
[0036] FIG. 2 illustrates the details of a comb developed in
accordance with the present invention; and
[0037] FIG. 3 illustrates the support plate developed for
supporting the present cassette.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The invention relates to the field of electrophoresis, and
more particularly to a cassette suitable therefor. It is to be
assumed that the gel used as the electrophoretic medium is
preferably an acrylamide (or polyacrylamide) gel, whether
cross-linked or not. However, other conventional and well known
electrophoretic media such as agarose gel or starch gel, can be
used. Polyacrylamide gel is particularly preferred because it is
transparent, electrically neutral, and can be made in various pore
sizes. Other co-monomers well known in the field include
N,N'-methylenebisacrylamide, N,N-bisacrylylcystamine,
N,N'-(1,2-dihydroxyethylene)bisacrylamide,
N,N'-diallyl-tartardiamide, and the like.
[0039] The drawings provided herewith are for the sole purpose of
illustrating preferred embodiments of the invention, and shall not
be considered as limiting the scope thereof.
[0040] Referring to drawings, FIG. 1 illustrates a cassette
assembly 10 and a support 12 therefor. Cassette 10 comprises a top
plate 14 and a reservoir plate 16 each of a substantially square
shape and having their four edges sealed, either with an adhesive
layer 11 inserted therebetween, or with the help of any other
compatible sealing means such as glue, ultrasonic welding, tape
etc. The structure of layer 11 is complementary with that of both
plates 14 and 16.
[0041] Plates 14 and 16 are preferably made of a chemically and
electrically inert material having the desired degree of rigidity
to support and protect the gel during casting thereof, as well as
shipping and handling operations. A thermoplastic "thermoformable"
material is most preferred because the plates can be produced
commercially via sheet thermoforming, which is quick, reliable and
relatively cheap. Preferred thermoplastic materials suitable for
the purposes of the invention include any electrically and
chemically inert thermoplastic material that can be easily and
economically thermoformed. Most preferred examples are polystyrene,
high density polyethylene (HDPE), low density polyethylene (LDPE),
linear low density polyethylene (LLDPE), polyethylene terephtalate
(PET), glycol-modified PET, polyethylene naphthalate, polyvinyl
chloride (PVC), polyvinylidene chloride (PVDC), polycarbonate,
PMMA, polyvinylacetate (PVA), ethylene vinylacetate (EVA),
polypropylene, polyesters, cellulose acetates, polyamides such as
nylon.TM., and copolymers thereof. Preferably, both plates 14 and
16 are made of the same material for compatibility purposes. In
addition, at least reservoir plate 16 should be transparent, but it
is preferred that both plates 14 and 16 be transparent.
[0042] Reservoir plate 16 comprises a series of reservoirs 18 for
receiving a corresponding series of teeth 20 of comb 22. Top plate
14 has a complementary structure, i.e., a series of openings 24,
that allow the passage therethrough of the plurality of teeth 20
for engagement into reservoirs 18. Reservoir plate 16 further
comprises a series of slots 26 aligned with the series of
reservoirs 18, and of substantially the same width. During filling,
shipping and handling operations, these slots are sealed with a
removable sealing strip 28 that will be removed before placing
cassette 10 in the electrophoresis device. In an alternate
embodiment, it has been found that the series of slots 26 can be
replaced with slots having a smaller width but being present in a
greater number, i.e, preferably twice the number of slots 26, with
the same end result.
[0043] Comb 22 comprises an aperture or inlet 30 extending
therethrough substantially perpendicularly to its longitudinal
section, and aligned with a tooth 32, the latter comprising a
longitudinal recess 34 shown in phantom lines in FIG. 2 and serving
as an outlet. After engagement of teeth 20 into the series of
reservoirs 18, an electrophoretic medium is injected into cassette
10 through aperture 30 and recess 34, as indicated by arrow 36. The
flow of electrophoretic medium inside cassette 10 is also indicated
by arrows 31 and 33. To ensure complete and proper filling of
cassette 10, as well as minimizing air bubbles, a slight excess of
electrophoretic medium must be injected. Such excess is discharged
out of cassette 10 through a longitudinal recess 38 provided in
each tooth 18. The flow of discharge is indicated by arrow 44.
Recess 38 is located on the side of a tooth 20 that is opposite to
the side of tooth 32 comprising recess 34. Each tooth 20 further
comprises a pair of grooves 40 and 42, the depth of which being
much smaller than that of recess 38, and arranged to form a V. The
purpose of these grooves is mainly to facilitate gel separation
from comb 22 upon removal thereof after completion of
polymerization of the electrophoretic medium, although they may
also be useful for discharge of excess of gel. Grooves 40 and 42
allow a clean separation of comb 22 from the gel, thus leaving a
lower surface of reservoir 18 containing the medium substantially
similar and even in each reservoir 18.
[0044] During the electrophoretic medium casting process, the
medium is poured into cassette 10 through opening 30 of comb 22,
and allowed to solidify. Preferably, the cassette is held in a
manner such that plates 14 and 16 are kept substantially parallel
to facilitate the filling of the cassette. Plates 14 and 16 can be
kept substantially parallel by, for example, applying a tension on
each side thereof to stretch its position, or a "non-sticky" glue
is applied on the external surface of the plates, so that the
latter can be removably "stuck" during injection of the
electrophoretic medium therebetween. Alternately, a vacuum can be
applied both outside and inside the cassette, i.e., inside for
drawing the gel inside the cassette, and outside for maintaining
the plates substantially parallel. A combination of vacuum outside
and positive pressure inside the cassette may also be used. The
polymerization process begins after an excess of medium has poured
out of each recess 38, confirming complete filling of cassette 10.
This method therefore substantially eliminates air bubbles from
cassette 10. Once polymerization is complete, cassette 10 is stored
appropriately in a conventional manner.
[0045] Comb 22 is preferably removed only minutes prior to the use
of the cassette, or immediately after complete polymerization of
the gel, prior to storage, if the reservoirs 18 are well preserved
from dehydration. At that point, it is slowly pulled out of the
cassette, and each reservoir 18 is thereafter filled with an
appropriate volume of a sample to be electrophoresed.
[0046] It is well known that in operation, the temperature of the
electrophoresis gel increases. It is also well known that the
temperature will be higher towards the middle of the cassette than
on the sides thereof. As a result, the migration front of the
products to be separated is altered, and erroneous interpretation
might occur. A novel support plate has therefore been developed to
overcome these problems, as well as for providing a proper profile
maintenance, i.e., sufficient rigidity of the thin walls of the
cassette, and facilitating installation of the cassette into an
operational position in a conventional electrophoresis
apparatus.
[0047] Support plate 12 comprises a frame 46 adapted to receive
therein cassette 10, and comprising a surface 48 with a plurality
of longitudinal recesses 50, which can be of any shape and size.
Openings 52 and 54 are cut within the plate to define a free space
substantially corresponding in size to reservoir 18 and slots 26.
When cassette 10 is placed onto support 12, it lies directly onto
ridges 56 of plate 12, thus forming a series of channels between
recesses 50 and a surface of cassette 10 for circulation of the
buffer solution therein (flow indicated by arrows 51), and thus
helping dispersing heat generated within the cassette. As
illustrated, each recess 50 is preferably aligned with a reservoir
18 and a slot 26, to ensure that the temperature of the migrating
product and the gel is substantially the same, whether the
reservoir is near the side or the middle of the cassette. It has
however been found that such alignment is not mandatory. The
critical element is that some buffer solution is allowed to
circulate between the support plate and the cassette to "extract"
heat from the latter. Support 12 can be made of any suitably rigid
material, but is preferably made of a heat conducting material, so
that heat is also extracted from ridges 56 that are in direct
contact with the surface of cassette 10 lying thereon, and
dispersed within the structure of the support. Cassette 10 can be
maintained in place in plate 12 with the help of a couple of
retainer plates 58.
[0048] With respect to the problem of interference of the
polymerization process caused by the thermoplastic material of the
cassette, it has been found that by combining a powerful initiator
generating more free radicals with an appropriate "sticking" agent
in the gel composition, there is no longer a need to apply a costly
protective layer over the thermoplastic surfaces. Examples of such
initiators include ammonium persulfate and
N,N,N,N-tetramethylethylenediamine (TEMED);
4-dimethylaminopropionitrile; 1-hydroxycyclohexyl phenyl ketone;
2,2-diethoxy-acetophenone; 2,2-dimethoxy-2-phenylacetophenone;
2',4'-dimethoxy-acetophenone; 2-hydroxy-2-methyl-1-propiophenone;
2-hydroxy-2-methyl-1-phenylpropan-1-one, and mixtures thereof.
These strong initiators allow a substantially complete
polymerization of the gel. However, the resulting polymerized gel
does not stick to the plastic surface, which is critical,
particularly in view of the fact that the cassette structure is
relatively flexible. Detachment or unsticking of the polymerized
electrophoretic medium from the cassette inner surfaces may lead to
the introduction of undesirable air bubbles between the plastic
surface and the gel, and may also cause irregularities in the
medium structure, thus severely impairing the efficiency of the
cassette. Surprisingly, it has been found that by adding to the gel
composition a small amount of an adhesive compound is sufficient to
allow the gel to adequately stick onto the plastic surface. The
adhesive compound preferably corresponds to that used for coating
the inner surfaces of currently available thermoplastic cassettes
for the same purpose. However, the costs associated with the
processing and coating of such a layer on the inner surfaces of the
cassette are significant. On the other hand, in the present
invention, all one has to do is to add a sufficient amount of the
said adhesive compound into the gel composition to be injected into
the cassette to achieve the same result. Not only is the procedure
more simple, but the amount of adhesive compound required is
smaller. Suitable adhesive compounds include polysilazanes or
tetra-substituted silicon derivatives. The substituents can be the
same or different, and include a straight or branched alkyl,
alkoxy, ketone, ester or amide each comprising from 1 to 8 carbon
atoms, or an amino, halogen, cyano or hydroxy. Preferred adhesives
are alkyl alkoxy silane derivatives. Most preferred adhesives
include Silane A-174, methacryloxytrimethoxysilylpropane,
3-(trimethoxysilyl)propyl methacrylate,
3-methacryloxypropyltrimethoxysilane, MEMO, DYNASYLAN MEMO, and
.gamma.-methacryloxypropyltrimethoxysilane.
[0049] The thickness of plates 14 and 16 should be sufficient to be
rigid enough for operation in an electrophoresis system. For
economical purposes, it has been found that it is not necessary to
exceed a thickness of about {fraction (40/1000)}.
[0050] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications, and this application is intended
to cover any variations, uses or adaptations of the invention
following, in general, the principles of the invention, and
including such departures from the present description as come
within known or customary practice within the art to which the
invention pertains, and as may be applied to the essential features
hereinbefore set forth, and as follows in the scope of the appended
claims.
* * * * *