U.S. patent number 3,635,808 [Application Number 04/877,378] was granted by the patent office on 1972-01-18 for method and apparatus for forming electrophoresis apparatus and the like.
Invention is credited to Franklin R. Elevitch.
United States Patent |
3,635,808 |
Elevitch |
January 18, 1972 |
METHOD AND APPARATUS FOR FORMING ELECTROPHORESIS APPARATUS AND THE
LIKE
Abstract
Thin film apparatus for electrophoresis, chemical analyses and
the like is provided, the film being an aqueous gel such as agarose
gel. Improvements in the manufacture of such apparatus are provided
such as the use of paraffin wax on the cover part of the apparatus
to facilitate separation of the film of gel from the cover. Also
continuous and semicontinuous methods of production are
provided.
Inventors: |
Elevitch; Franklin R. (Palo
Alto, CA) |
Family
ID: |
27416314 |
Appl.
No.: |
04/877,378 |
Filed: |
November 17, 1969 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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300341 |
Aug 6, 1966 |
|
|
|
|
579089 |
Sep 13, 1966 |
|
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|
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664133 |
Aug 29, 1967 |
3479265 |
Nov 18, 1969 |
|
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Current U.S.
Class: |
204/620; 422/940;
422/941; 422/945; 422/944 |
Current CPC
Class: |
B01L
3/508 (20130101); B01L 3/505 (20130101); B01L
3/545 (20130101); G01N 27/44756 (20130101); G01N
27/447 (20130101); G01N 27/44747 (20130101); G01N
27/44704 (20130101); G01N 27/44721 (20130101) |
Current International
Class: |
B01L
3/00 (20060101); G01N 27/447 (20060101); B01k
005/00 () |
Field of
Search: |
;204/18G,18S,18R,299 |
Other References
hoechst, "Partigen Behringwerke Immunodiffusion Plates," available
from Certified Blood Donor Service, Inc., Woodbury, NY, 11797 .
Smithies, "An Improved Procedure of Starch Gel Electrophoresis,"
Jrnl. of Biochem., Vol. 71, No. 3, (1955), pp. 585-587.
|
Primary Examiner: Mack; John H.
Assistant Examiner: Prescott; A. C.
Parent Case Text
DESCRIPTION
This application is a continuation-in-part of the following
copending applications: Ser. No. 300,341 filed Aug. 6, 1963, now
abandoned; Ser. No. 579,089 filed Sept. 13, 1966, now abandoned and
Ser. No. 664,133 filed Aug. 29, 1967, now U.S. Pat. No. 3,479,265
granted Nov. 18, 1969.
Claims
I claim:
1. A method of forming apparatus adapted for gel diffusion analysis
such as electrophoresis which comprises:,
providing a base having a surface which is adherent to aqueous,
permeable gels,
applying a layer of such a gel to said adherent surface, and
applying a cover to said layer of gel with one surface in contact
with said layer of gel,
the contacting surface of said cover being coated with a normally
solid, thermoplastic essentially hydrocarbon, hydrophobic material
which is nonadherent to said gel.
2. The method of claim 1 wherein said hydrocarbon material is
paraffin wax.
3. The method of claim 2 wherein said cover is formed on its
contacting surface with well-forming projections that project into
the layer of gel and form sample-receiving wells therein.
4. The method of claim 2 wherein said layer of gel does not exceed
about 1 millimeter in thickness.
5. The method of claim 2 wherein the paraffin wax is applied in
solution in a volatile solvent.
6. The method of claim 5 wherein the applied coating of wax, after
evaporation of solvent, is heat glazed.
7. Apparatus adapted for gel diffusion analysis such as
electrophoresis comprising:
a. an impermeable base member,
b. a layer of permeable gel having a surface in adherent contact
with said base member,
c. a cover member with an inner surface uniformly coated with a
solid thermoplastic, essentially hydrocarbon, hydrophobic material
to which the gel is nonadherent, the coated inner surface of said
cover member being in contact with the other surface of said layer
of gel.
8. The apparatus of claim 7 wherein such coating material is
paraffin wax.
9. The apparatus of claim 8 wherein said cover member is formed
with well-forming projections which project into the gel to form
sample-receiving wells.
10. The apparatus of claim 8 wherein said layer of gel does not
exceed about 1 millimeter in thickness.
Description
In my copending applications referred to above thin film methods
and apparatus for conducting electrophoresis (and subsequent
fluorometry or densitometry) and for conducting radial diffusion
operations are described.
It is an object of the present invention to provide improvements in
such apparatus and methods.
It is a particular object of the present invention to provide
improved separation of the cover of the apparatus from the base so
that none or a negligible quantity of the film of aqueous gel
adheres to the cover.
It is a further object of the present invention to provide
improvements in the process of manufacturing such apparatus.
Other objects of the invention will be apparent from the ensuing
description and the appended claims.
Certain forms of the invention are illustrated by way of example in
the accompanying drawings in which:
FIG. 1 is a perspective view if one form of apparatus in accordance
with the present invention showing the cover and the base;
FIG. 2 is a section taken along the line 2--2 of FIG. 1 but with
the base applied to the cover;
FIG. 3 is a perspective view of the base of the apparatus of FIG. 1
separated from the cover and with a film of gel adhering to it;
FIG. 4 is a cross section taken along the staggered line 4--4 of
FIG. 3;
FIG. 5 is a diagrammatic drawing of a semicontinuous method for
manufacturing apparatus such that as that shown in FIG. 1;
FIG. 6 is a view in vertical section through a thin film device as
made by the method of FIG. 5;
FIG. 7 is a diagrammatic drawing showing a continuous method of
manufacturing thin film devices;
FIG. 8 is a view taken along the line 9--9 of FIG. 7 showing in
elevation the roller used to fabricate thin film devices;
FIG. 9 is a fragmentary view showing an alternative to the
apparatus of FIGS. 7 and 8;
FIG. 10 is a view in vertical section of a stack of devices in
accordance with the invention;
FIG. 11 is a diagrammatic drawing showing a continuous method of
analysis employing the thin film technique of my invention.
In the apparatus of my invention the film of permeable gel which
provides the medium for migration of specimens, reagents and the
like is an aqueous gel and it is preferably a thin film, usually
not more than 1 millimeter thick and preferably not more than about
0.5 millimeter thick. However, in some embodiments of the invention
the film of gel may be thicker. This film is permeable to reagents
such as those mentioned hereinafter and to biological materials
such as proteins, blood serum, etc., such that the selected
reagents, specimens, etc., will migrate by capillary effect or
under an applied voltage.
The thin films of gel used in accordance with my invention are also
preferably uniform in thickness, and preferably do not depart by
more than about 10 percent from the average thickness.
The thinness and uniformity of these films are highly advantageous.
The gel thinness permits rapid migration under an applied voltage
of 10-15 volts per centimeter or more without the need for applied
cooling and without destructive heat buildup which would dry the
gel or denature the material migrating into the film, such as
proteins. This rapid migration without elaborate cooling equipment
and without destructive heat buildup is advantageous in
electrophoresis because it permits the use of high voltages with
attendant high speed of separation, and without applied cooling,
and it also permits the use of inexpensive equipment which can be
supplied to the customer ready for use and which is inexpensive
enough to discard after a single use. In the radial diffusion
technique thin films are advantageous because they allow the use of
minimum quantities of expensive reagents such as antisera and they
also provide sharp end points. In all cases uniformity of film
thickness is advantageous because results are more reliable.
Advantages of thin films of gel have been noted heretofore. See
Wadsworth, "A Slide Microtechnique for the Analysis of Immune
Precipitates in Gel, " Int. Arch. Allergy, vol. 10, pp. 355-360
(1957). However, to my knowledge, prior to my inventions practical
apparatus employing thin films of gel which lends itself to
manufacture in a form ready for use, which can be stored for
extended periods of time without deterioration, which is simple to
use and which is sufficiently inexpensive that it can be used once
and discarded, has not been available.
In FIGS. 1 and 2 one form of apparatus is shown which provides
these advantages. Referring now to FIGS. 1 and 2, a complete mold
is there shown which comprises a cover 10 and a base 11 which may
be made of the same or different materials such as polystyrene,
polyethylene, polypropylene, etc. Polystyrene is a preferred
material because of its superior machining and fabrication
qualities and because it is transparent and nonfluorescent.
Nonfluorescence is of particular importance in applications where
fluoremetry is used because a fluorescent material is incompatible
with this technique. For example, where the separated proteins are
stained with a dye which fluoresces, background fluorescence of the
material of the base would be incompatible. Polyethylene and
polypropylene meet these requirements but their machining and/or
fabrication qualities, although acceptable, are not as good as
those of polystyrene. Other materials of construction may be used.
The cover 10 preferably has substantial thickness and is rigid
while the base 11 is preferably thin and flexible. Transparent
materials such as polystyrene, polypropylene and polyethylene are
preferred although where reflectance methods of fluorometry are
used an opaque material may be used. However, even in those cases
transparent materials of construction are preferred because, among
other things, they permit visual inspection to detect bubbles,
voids, etc.
The base 11 preferably has a flat inner surface. The cover 10 is
provided with a rim formed by end walls 12 and sidewalls 13 which
define a mold cavity 14 and which determine the depth of the mold
cavity and therefore the thickness of the resulting film of gel.
The rim 12, 13 provides a face 16 to which a pressure sensitive
adhesive is applied as explained below. The inner surface of the
cover 10 is formed with risers 19 and well-forming projections 24a
and 24b. As shown in FIG. 2, the risers 19 have the same height as
the rim 12, 13 and they are intended to contact the base 11 when
the apparatus is assembled. The risers 19 and the rim 12, 13
determine the depth of the mold cavity and therefore the thickness
of the film of gel. They are formed with accuracy for this purpose
so that the film of gel has a uniform thickness. Preferably the
mold cavity depth and therefore the film thickness are less than 1
mm., preferably about 0.3-0.5 mm.
The well-forming projections 24a and 24b, as appear in DIG. 2,
preferably terminate short of the base 11 so that the resulting
sample-receiving wells do not extend to the base. This is shown in
FIG. 4 and it is advantageous because, if the wells extend through
the base, undercutting of solution placed in the wells may occur
with resulting fuzzy or ambiguous results. In FIGS. 3 and 4 a layer
of gel 28 is shown adhering to the base 11, such film being formed
with sample-receiving wells 29a and 29b and with channels 31 formed
by the well forming projections 24a and 24b and the risers 19,
respectively. The cover 10 is provided with filling and venting
openings 26. Caps (not shown) may be provided to close the vents
but preferably the vents 26 are heat sealed to close them.
In FIG. 4 the cover 10 is shown in broken line, the purpose being
to illustrate how the projections 24a and 24b and the risers 19
remain within the wells 29a and 29b and the channels 31 until such
time as the base 11 with the adhering film of gel 28 is stripped
from the cover (see below). This is an important advantage of the
construction of my invention because the gel, being aqueous (about
90 percent water), will leak into the wells 29a, 29b, also into the
channels 31. Therefore, without the projections 24a, 24b and risers
19, the wells and channels would be filled and their integrity
would be destroyed. By reason of the projections 24a, 24b and the
risers 19 their integrity is preserved during long periods of
storage, and by reason of the edge sealing described below, the
integrity of the entire film against evaporation is also preserved
during long periods of storage.
As described in my copending applications above mentioned the gel
28 is adherent to the base 11 and is nonadherent to the cover 10
such that, when it is desired to separate the base it may be
stripped from the cover with the film of gel 28 adhering to the
base and completely separated from the cover, to which none or a
negligible quantity of the film adheres. A material of construction
may be selected for the cover which is nonadherent to the gel and
another material may be selected for the base which is adherent. An
example of nonadherent materials for the cover are various
polysiloxanes such as Silgard, which is a trademark of Dow Corning
Corporation which is described in a brochure of that company,
Bulletin 07-2713, dated March, 1968. It is described as a
transparent, room-temperature curing, solventless resin supplied as
a nearly colorless, free flowing, low viscosity fluid having a 100
percent silicone resin content. It is mixed at or shortly before
the time of use with a curing agent in the proportions of 1 part by
weight of curing agent and 10 parts by weight of resin. Other data
and information regarding this resin will be found in said
bulletin. Other room-temperature curing silicone resins may be used
instead of Silgard, which is further identified in the aforesaid
publication as Silgard 184. Examples of adherent materials for the
base are polystyrene, polyethylene and polypropylene. However, it
is preferred to rely upon a release agent to prevent adherence of
the gel to the cover 10, which may therefore, be made of the same
material as the base 11. As set forth in my copending application
Ser. No. 664,113 now U.S. Pat. No. 3,479,265, a silicone release
agent may be employed for this purpose.
In accordance with the present invention an improved type of
release agent and an improved method of applying the release agent
are provided for the cover 10. For this purpose a solution of
paraffin wax in a volatile solvent is applied to the cover 10 by
dipping, spraying or other means. A suitable wax, when the cover is
made of polystyrene having a softening point of about 90.degree.
C., is a refined paraffin wax having a melting point (AMP) of
128.degree./130.degree. F.; a color (Saybolt, ASTM D156) of +30; an
oil content (ASTM D721 )=0.3 percent; a penetration at 77.degree.
F. and 100.degree. F. (ASTM D134)=17 and 105, respectively; and a
viscosity at 180.degree. F. and 210.degree. F. (SSU, ASTM
D2161)=41.6 and 38.2, respectively. Any other paraffin wax, and in
fact any other thermoplastic, essentially hydrocarbon, hydrophobic
material may be used which will adhere to the cover, which is
nonadherent to an aqueous gel such as agarose gel, which is solid
at temperatures up to about 70.degree. C. and which melts
substantially below the melting point of the cover material. A
solution, e.g., in low boiling petroleum ether
(b.p.=36.degree.-40.degree. C.), is applied by any suitable means
as by dipping or spraying. It does not matter that the top portion
of the cover is also coated inasmuch as the amount wasted is not
great and does not interfere with subsequent use of apparatus. If
the solution is applied by dipping, the cover may be allowed to
drain. If the solution is applied by spraying, it is applied so as
to provide a uniform coating of wax on the surface.
Next, the cover is dried by air drying at ambient temperature, by
means of applied heat such as a current of hot air or by any other
suitable means. This leaves a very thin coating of wax on the inner
surface of the cover. This coating is uniform but contains small
voids. To eliminate these voids, the coating of wax is melted and
glazed by a mild application of heat, as by means of a current of
hot air, by holding over a hot plate or otherwise. The temperature
and time of heating are such that glazing of the wax coating occurs
but without damage to the material of which the cover 10 is
constructed. By this means the coating on the cover is made uniform
and voids are filled in by coalescence.
Before or after this wax coating operation, preferably afterwards,
a pressure sensitive adhesive such as a rubber cement is applied to
the face 16 of the rim 12,13 and/or to the corresponding marginal
area of the base 11. Preferably the pressure sensitive adhesive is
applied to the face 16 of the rim 12,13 of the cover 10. Also heat
sealing may be employed instead of adhesive.
Next the cover 10 and base 11 are assembled as shown in FIG. 2 and
sufficient pressure is applied, as by means of a platen press, to
adhere the base firmly to the cover. This pressure brings the face
16 of the rim 12,13 of cover 10 into firm, uniform contact with the
cover 11, and it also brings the risers 19 into firm contact with
the cover 11, so that the space between the cover 10 and base 11,
hence the thickness of the resulting film of gel is uniform. Then a
melted gel is introduced through one of the openings 26, air being
vented through the other opening. A suitable gel formulation
consists of 10 percent by weight of sucrose and 1 to 1.2 percent by
weight of agarose dissolved in an electrolyte solution. The
electrolyte solution may be an aqueous solution of sodium barbital
and HCl having a pH of 8.6. Many such aqueous gels and aqueous
electrolytes are well known. Other gelling agents such as starch,
agar, acrylamide, etc. may be used in place of agarose. The
formation of such gels is well known in the art. A gel formed in
accordance with the complete formulation given above is
advantageous because it is transparent and nonfluorescent and it
has good properties for migration of materials by capillary effect
and/or under a voltage gradient as in electrophoresis.
Preferably the gel is degassed before it is placed in the mold. It
has been found that, on occasion, the film of gel forms gas
bubbles, especially if the apparatus is stored in a hot climate or
in hot surroundings. Such degassing is readily accomplished by
placing the gel in a vacuum vessel, applying sufficient heat to
melt it and subjecting it to a vacuum of about 28 inches of mercury
for about 5-10 minutes. The gel may be stirred to accelerate
degassing.
Preferably also the gel has incorporated in it a very small amount,
e.g., about 0.05 to 0.2 percent by weight of a polyvinyl alcohol. A
suitable polyvinyl alcohol is a 99 percent hydrolyzed (i.e., 99
percent of the acetyl groups of polyvinyl acetate are removed by
hydrolysis), having a viscosity of 28-32 centipoises. This is used
as a 4 percent aqueous solution. I have found that the inclusion of
polyvinyl alcohol considerably improves the release properties of
the gel from the cover.
After the mold (which is in a press, see above) has been filled
with gel (which is supplied in warm, molten condition), as
indicated by an overflow from the venting spout 26, the caps 27 are
applied to the spouts 26 or the spouts 26 are heat sealed and,
after the gel has cooled and solidified, the apparatus is ready for
storage, shipment and use.
In use, as described in my copending application Ser. No. 664,113,
now U.S. Pat. No. 3,479,265, the base 11 is peeled from the cover
and carries with it a thin, uniform film of gel. This film of gel
is formed with troughs or channels 31 formed by the risers 19 and
sample-receiving wells 29a and 29b formed by the well-forming
projections 24a and 24b. By reason of retention of the cover 10 and
its projecting risers 19 and well-forming projections 24a and 24b
until the time of use the integrity of the troughs and wells is
preserved. Specimens are placed in the wells 29a and/or 29b. If
separation by electrophoresis is to be carried out, the base 11
with its adhering film of gel is placed in a suitable
electrophoresis apparatus and is subjected to a suitable voltage
gradient to effect separation of proteins. As described in my
copending application Ser. No. 664,113, now U.S. Pat. No.
3,479,265, by reason of the uniformity of the layer of gel, sharp
separations are produced and because of the thinness of the gel,
high-voltage gradients (e.g., 10 to 15 volts per centimeter) can be
applied without external cooling. Rapid separations are effected,
e.g., in one-half hour to 1 hour. The apparatus is then placed in
suitable analytical equipment, after suitable staining and other
processing. The analytical equipment may be a fluorometer or a
densitometer to measure quantitatively the separated proteins. If
the apparatus is to be used in the radial diffusion-precipitation
technique, the body of the gel will have been impregnated with a
suitable reactant such as an antiserum and specimens, e.g., blood
serum, will be placed in the sample receiving wells and will
diffuse outwardly and will form rings (which can be made visible or
more readily visible by staining) by reaction with antiserum.
Measurement of the diameters of these rings and of the diameters of
rings produced by control specimens of known material will provide
a quantitative measure of the material to be measured. Further
details will be found in my copending application Ser. No. 664,
113, now U.S. Pat. No. 3,479,265.
In the foregoing description, the film of gel is described as
having sample receiving wells formed in it by well forming
projections. In another embodiment of the invention the film of gel
is made without such wells, in which case the cover 10 is formed
without the projections 24a, 24b and if desired, without the risers
19. Specimens may be applied to the plain surface of the film of
gel and caused to migrate radially outwardly as in the radial
diffusion technique, or to migrate directionally as in
electrophoresis. Wells may be formed in the film after the cover 10
is removed, or the cover 10 may be formed with holes which are
sealed during storage, then opened at the time of use to permit
introduction of specimens. Alternatively, a template with holes in
it may be placed over the exposed film (after removal of the cover)
and specimens introduced through the holes.
In accordance with another embodiment of the invention, apparatus
such as that shown in FIGS. 1 and 2 may be made in a semicontinuous
way. Referring to FIG. 5 a continuous tape 40 supplied from a roll
(not shown) is provided, such tape being, e.g., polystyrene and
having the same thickness as the base 11 in FIG. 1, e.g., about
0.005 inch. This tape may be passed through a tank 41 filled with a
cleaning solvent such as isopropyl alcohol although such may be
unnecessary. The tape is guided around a guide roller 42 and is
dried by means of a current of hot air (not shown). The tape then
passes along a table 43 through a film-forming station A and pauses
at timed intervals at that station while a mold 44 is lowered into
the tape. The mold 44 has a rim which forms a mold cavity 46 having
the desired depth and horizontal shape and dimensions. The mold 44
is shown, for simplicity, without well forming projections and
risers such as 24a, 24b and 19 but it may be provided with them. A
rod 47 connected to a suitable operating mechanism (not shown)
lowers the mold from the raised position shown in broken lines to
the lowered operating position shown in solid lines in contact with
the tape. During this pause or dwell and while the mold is in the
firm contact with the tape, a gel is injected from a suitable
source (not shown) through one of the spouts 48. Air is vented
through the other spout along with excess gel. The gel is
introduced in liquid or semisolid condition and the dwell at
station A is long enough to permit such filling operation and to
allow the gel to solidify. If desired a current of cold air may be
applied to the mold 44 and/or to the under surface of the table 43,
or cooling liquid may be passed through passages (not shown) in the
mold and/or table to hasten solidification of the gel. The mold is
then tilted to the angular position shown in dotted lines to
release it from the solidified film of gel and it is then raised to
the horizontal position, also shown in dotted lines, to allow the
tape to move another increment. Preferably the interior of the mold
will have been coated with a suitable release agent unless the
material of construction is inherently nonadherent. Suitable
release agents are silicone release agents and paraffin wax as
described above. The tape 40 also pauses at an applicator station B
where a mold cover 10 identical with that shown at 10 in FIG. 1 may
be applied. This application may be manual or it may be done
automatically by a suitable mechanism (not shown) during a swell in
the tape movement. The mold cover will have been coated with
paraffin wax, or if desired with some other release agent, as
described above. Also the face 16 of the rim of the cover (and/or a
corresponding marginal area of the tape) will have been coated with
a pressure sensitive adhesive. As an aid, especially where manual
application of the mold cover is employed, the upper face of the
tape 40 may be printed at intervals with an outline of the rim of
the mold.
Alternatively, and as shown in FIG. 5, the film of gel 50 formed at
station A may be coated with a material such as Silgard. This may
be done by spraying or by flowing the material onto the film of
gel. The solvent, if one is used, is allowed to evaporate, such
evaporation being accelerated if desired by a current of hot air,
exposure to a heat lamp or other suitable means. The coating
material is selected to harden, as by catalytic polymerization in
the case of Silgard. Downstream from station B the tape is severed
by a knife (not shown) to produce a complete package 55 (FIG. 6)
composed of a base 56 (the severed portion of tape 40), a thin film
of permeable aqueous gel 50 and a cover 57 formed of the coating
material applied at station B and which laps over the edges at 57a
around the entire periphery of the film 50. This cover also extends
into wells 59 and channels 60 formed in the film by the mold 44.
When it is desired to use the apparatus 55, a segment of the
overlap 57a is lifted (being pried loose with a blade if necessary)
and is stripped from the film 50, which is then ready for use. As
in the case of the cover 10 in FIGS. 1, 2 and 4, the cover 57, by
projecting into wells 59, etc. preserves their integrity during
storage.
Referring now to FIGS. 7 and 8 equipment is there shown for
continuous fabrication of apparatus of the present invention. This
equipment comprises a continuous tape 40, a tank 41 and solvent 42
and a guide roller 42a as in FIG. 5. A block 70 is shown which is
formed with passages 70a for circulation of cooling fluid. The
block 70 has a concave portion at 71 and a convex portion at 72. A
roller is provided at 73 which is nested in the concave portion 71
of the block 70 and at the lower end of a sloping portion 74. An
applicator such as a nozzle is provided at 75. Referring more
particularly to FIG. 8, at opposite sides of the sloping portion 74
of the block 70 and at opposite ends of the roller 73 retainer
plates 76 are provided. The roller 73 is provided with trunnions 77
by which the roller is rotatably supported in a suitable framework
(not shown). The roller 73 is provided with sprocket teeth 78 which
are intended to engage mating holes in the tape 40 so that the tape
moves synchronously with the rotary motion of the roller 73.
Alternatively, engagement of the tape and roller may be frictional
and without a sprocket arrangement. The roller 73 is shown formed
with mold units 79, each of which has a depth equal to the desired
thickness of the film of gel, that is preferably not more than 1
mm. and most preferably about one-half mm. or less. Each mold unit
comprises ribs or risers 80 and well-forming projections 81. The
ribs 80 extend to the periphery of the roller and therefore contact
the tape 40. The well-forming projections 81 preferably terminate
short of the periphery of the roller and therefore do not contact
the tape 40. Any desired number of mold units 79 may be provided,
four being shown on one face of the roller in FIG. 8, there being
four on the other face, although any number desired may be
used.
In operation the tape 40 is moved continuously through the
apparatus by reason of engagement of sprocket 78 with holes in the
tape (or by frictional engagement). Such movement is preferably at
a uniform rate such that liquid gel material may be supplied at a
uniform rate by means of the applicator 75 to maintain an adequate
pool of liquid or semisolid gel in the well formed by the sloping
section 74 of block 70 and the retainer plates 76. If need be, heat
may be applied to this pool to maintain it in adequately fluid
condition. As each mold unit 79 rotates into contact with this pool
of gel it will be filled with gel and will rotate into the cooled
sector of its cycle. Cold fluid circulating through the passages
70a in the block 70 and, if desired, through similar passages (not
shown) in the roller 73 acts to solidify the liquid gel thus
trapped in each mold unit 79. During transit along the concave
portion 71 of the block 70 this trapped body of gel solidifies.
There results from this mode of operation a multiplicity of films
80 of solidified gel each imprinted with wells and troughs formed
by the well-forming projections 81 and the ribs 80. The convex
portion 72 of the block 70 provides a reverse bend which assists in
separation of each film of gel 80 from the roller. These films of
gel, supported on the tape 40, pass along a table 81 and, at an
appropriate point, a cover such as that shown in FIG. 1 is applied
manually or automatically by suitable means (not shown) or,
preferably, as shown in the FIG. 7, the film is coated at 82 with a
material as described above in connection with FIG. 5. Subsequently
a knife 83 is used to sever the tape 40, resulting in the
production of individual packages such as that shown at 55 in FIG.
6.
In FIG. 8 the cylinder 73 is shown with longitudinal segments 85
separating the mold units 79. Alternatively, these segments may be
omitted, i.e., the dotted lines 86a in FIG. 8 represent
continuations of the sidewalls 86 of the respective mold units 79.
In such case, the cylinder 73 is formed with one or more (two being
indicated in FIG. 8) continuous circumferential recesses with ribs
80 and well forming ribs 81 located therein. As a result, the
product issuing from the cylinder at the convex portion 72 of block
70 comprises not only a continuous tape 40 but also a continuous
film (or two or more continuous films) of gel with wells (and such
other impressions as desired formed therein at intervals. Covers
may be applied as described above, and in the case of a coating as
shown in FIG. 7, this cover may be continuous. The continuous,
tapelike laminar product may be severed into segments as and when
desired, or it may be wound up in continuous form into a roll for
subsequent use, severing, etc.
Further, wells and troughs need not be formed in the film, whether
formed in separate segments as shown in FIG. 7, or as a continuous
tape as just described. In such case, if sample receiving wells are
needed, they may be formed subsequently. Also specimens may be
applied to plain film, i.e., film formed without an imprinted
pattern of wells, etc. in the manner described above.
Referring now to FIG. 9, an alternative to the cylinder 73 of FIGS.
7 and 8 is shown. This alternative, generally designated as 100,
comprises an endless member 101 extending about wheels or sprockets
102 and 103 (with intermediate wheels or sprockets if needed). A
table 104 has a downwardly sloping section 105 at the input end
(the lower reach of the member 101 moving from left to right), and
an upwardly sloping section 106 at the other end. The endless
member is formed with separate mold sections like sections 79, or
with a continuous mold section, with or without well-forming
projections as at 81 and risers as at 80 as desired. That is to
say, the endless member 101 is, in effect, a transformed cylinder,
lengthened out and having an operative, mold forming configuration.
Tape is shown at 40, a supply nozzle at 75 and film 80a on the tape
is also shown. An advantage of the apparatus of FIG. 9 is that it
provides an elongated cooling path wherein the molding element
(endless member 101) has sufficient length to allow solidification
of the film of gel. Cooling fluid may be passed through the endless
member 101 and/or the table 104 and/or a current of cold air may be
applied to hasten solidification of the gel.
In those embodiments of the invention in which the film is plain
and is not imprinted with wells and the like whose integrity must
be preserved, the film of gel, after being formed, may be covered
with a nonadherent sheet material such as polyethylene film coated
with a release agent such as silicone release agent or paraffin
wax, and the resulting packages may be stacked one on top of the
other. Edge sealing to prevent evaporation of the gel may be
provided by overlap of the overlying sheet material, by heat
sealing such overlap, etc., or by packaging the film in a tight
container which seals the exposed edges.
A packaged product formed in this manner is shown in FIG. 10. The
package is generally designated as 110 and it comprises a bottom
closure 112, sidewalls 113 and a top closure 114. The parts 112,
113 and 114 may be integral and they may be made of polyethylene,
polystyrene or any other suitable material. Within this container
are a multiplicity of units each comprising a base 115 and a film
of permeable gel 116. The top surface of each base is adherent to
the gel and the bottom surface is nonadherent to the gel. For
example the base members 115 may be made of polystyrene, which is
adherent and the bottom surface of each polystyrene base may be
coated with a release agent such as a silicone release agent or
paraffin wax. Therefore, when the package is opened, the top base
member 115 may be stripped from the stack with the superimposed
film of gel adhering to it but the layer of gel beneath being
nonadherent. The arrangement shown in FIG. 10 with each base 115
beneath and the adhering film of gel above, may be reversed, i.e.,
the top surface of each base 115 may be made nonadherent and the
bottom surface may be made adherent. In effect, in this variant,
the package 110 in FIG. 10 is merely inverted so that 112 becomes
the top and 114 becomes the bottom.
Another embodiment of the invention is shown in FIG. 11. In this
embodiment a length of tape 120 is provided which travels
continuously or by increments along a table 121 in the direction
shown. Adhering to the tape 120 is a film 121 of permeable aqueous
gel. This gel, which may be as described above, may be applied in
any manner, e.g., by brushing, squirting, spraying or dipping. It
is, preferably, a thin film, preferably not more than about 0.3-0.5
mm. and it is uniform in thickness. Such uniformity can be achieved
by various means, e.g., by passing the tape beneath a roller or a
doctor blade.
The layer of gel 121 may, if desired, have sample receiving wells
formed in it such as the wells 29a and 29b in FIGS. 3 and 4.
However, there need be no such wells.
The film of gel 121 may be uniformly impregnated with a reagent,
e.g., by premixing the gel and reagent before applying it to the
tape 120. The tape is moved beneath pipette 122 which applies a
measured amount of the unknown. If the tape is moved slowly, or if
the pipette is caused to undergo a cycle in which it moves
forwardly with and at the rate as the tape and then quickly
rearwardly to its starting position, the tape may move
continuously. Alternatively the tape may be moved by increments and
the unknown added during each pause between increments of forward
movement.
Then the film 121 may be processed (after it has travelled a
sufficient distance or has paused long enough for the reaction
between reagent and unknown to be completed) in any suitable
manner. For example, in the analysis of serum protein, the serum
protein is applied by the pipette 122 and any suitable dyestuff
such as 8-anilino naphthalene-1-sulfonic acid is applied by means
of a nozzle 123. The selected dyestuff is one like 8-anilino
napthalene-1-sulfonic acid which will attach itself to the protein
undergoing analysis and in so doing will become fluorescent in
proportion to the amount of protein. By this means the amount of
protein (the reagent dyestuff being added in excess) can be
determined by fluorometry. Other dyestuffs than 8-anilino
napthalene-1-sulfonic acid may, of course, be used provided they
function in this manner, i.e., they are nonfluorescent but attach
themselves to protein and become fluorescent in proportion to the
amount of protein-dyestuff complex. In the case of 8-anilino
naphthalene-1-sulfonic acid, it is preferably used in the form of
an acid solution e.g., 30 mg. of the dyestuff in 100 ml. of 5-10
percent aqueous acetic acid solution.
If desired a fixing step may be interposed between pipette 122 and
nozzle 123 to fix the protein, e.g., a dilute aqueous solution of
formaldehyde and perchloric acid may be sprayed onto the area to
which serum protein has been applied.
After the reagent has been applied by nozzle 123, after a fixing
step (if any) has been carried out and after such period of time as
is required for reaction of reagent with the protein to be
completed, the tape is passed through a fluorometer 124 which may
be provided with a readout to record the value of the determination
on a printed tape and/or on a screen.
8-anilino naphthalene-1-sulfonic acid is unstable in aqueous
solution if held for any great length of time; it should be used
when freshly prepared. This leads to another embodiment of the
invention, as follows: A film is provided as at 121 is FIG. 11, or
in any other form in accordance with the present invention.
8-anilino naphthalene-1-sulfonic acid is stable for long periods of
time if it is dry. The film 121 is impregnated with this reagent
and is then dried, e.g., by application of mild heat and/or vacuum.
The reagent is reactivated when moistened, which may be
accomplished by dipping the film in or spraying it with water just
before use. In many cases the water in the sample of unknown, e.g.,
blood serum, will serve the same purpose. In general, permeable
films, preferably but not necessarily made in accordance with the
present invention, may be impregnated with a labile reagent which
is stable in anhydrous environment but which is unstable in the
presence of moisture, and this film may be dried, stored, shipped
and delivered in dry form; and reactivated by adding moisture at or
just before the time of use.
Rather than using a tape, a disc formed of the same material may be
employed having an adhering film of gel, and this disc may be
rotated in a turret type of apparatus wherein reagent and/or
unknown may be applied at different points and a sector of the disc
may rotate through a fluorometer.
In the tape and disc embodiments of the invention illustrated by
FIG. 11 the extreme thinness and uniformity of the film of gel are
advantageous not only for the reasons previously stated but because
the steps involved, moistening, drying, curing, etc. can be
accomplished very quickly. If thick layers of gel are employed, the
time required for these steps may be prohibitive.
In general optimum thickness and optimum uniformity can be
determined by criteria such as those mentioned immediately above
and elsewhere above. I have found that thickness less than about 1
mm. should be used, preferably not more than about 0.5 mm. and that
departures from average thickness preferably do not exceed about 10
percent, most advantageously not by more than about 7 percent.
It will therefore be apparent that novel and very useful methods
and apparatus have been provided.
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